Application of recombinant DNA techniques

Experimental approaches that have afforded major insights to the processes described in this chapter include (1) use of yeast mutants (2) application of recombinant DNA techniques (eg, mutating or eliminating particular sequences in proteins, or fusing new sequences onto them and (3) development of in vitro... 

The application of recombinant DNA techniques to the study of neuropeptide biosynthesis is essential because these techniques facilitate structural analyses and evolutionary studies, clarify biosynthetic pathways, provide the necessary background and probes for analysis of synthesis regulation at the levels of mRNA and gene transcription and assist in classical genetic experimentation. Examples of these benefits are cited and problems encountered in insect systems are discussed. 

This chapter aims to describe some essential genetic manipulation techniques and illustrate, with some key examples, their use for the generation of recombinant pharmaceutical drugs. Applications of recombinant DNA techniques in the diagnosis of diseases also will be covered. 

New methods in biotechnology such as plant tissue culture, the application of recombinant DNA-techniques to plants and the biotransformation of oleo-chemlcals by immobilized enzymes and microorganisms hold promise to a)... 

Aside from these relatively direct applications of site-directed mutagenesis, combination of recombinant DNA techniques with other experimental strategies will no doubt prove to be of increasing importance. If the gene of interest can be expressed with sufficient efficiency in auxotrophs, then proteins in which selected amino acids are isotopically enriched [12] may be produced to increase the sensitivity and selectivity of magnetic resonance techniques. Alternatively, amino acid analogues that are recognized as substrates by aminoacyl tRNA synthetases may be incorporated randomly in place of the true substrate amino... 

A further important aspect is the feasibility of whole cell biotransformations. Whole cell biotransformations show a lot of advantages as compared to isolated enzymes, such as the improved stability of enzymes. If both, producing and regenerating enzymes, are available in one single strain, no addition of expensive cofactor is necessary because the intracellular cofactor pool can be utilized. Whole cell biotransformations are therefore very promising for technical applications, and making these conversions an intensively studied subject in the last years. The use of recombinant DNA techniques offers many possibilities to create capable systems. This chapter describes the most important whole cell biotransformations developed in the past as well as relevant processes with small-scale and technical application. 

Most of these enzymatic ways to regenerate nicotinamide cofactors have been successfully established in a multitude of synthetic processes. The introduction of organic-water two-phase systems seems to be a useful method to overcome problems with the low solubility of many organic substrates. Bioconversions using whole cells have been well investigated because they show some decisive advantages like increased enzyme stability. The use of recombinant DNA techniques offers a wide field of application. Tailor-made cells can be created to perform whole cell biotransformations efficiently. 

There are a limited number of issued patents and published patent applications relating to the use of recombinant DNA techniques in higher plants or in organisms which infect or otherwise are associated with them. It is expected that with the Hibberd decision, increased use will be made of the utility patent system to obtain coverage for plants per se, despite the alternatives provided by the Plant Patent Act and by the PVPA. 

However, when considering the use of probiotic strains as functional starters or co-cultures, it is important that they do not enhance acidification and thereby reduce the shelf-life of the product, and that they do not have adverse effects on the aroma or taste of the product (Heller, 2001). Uncertainty still exists as to whether multifunctional strains possessing all desirable metabolic features would result from modem isolation techniques and selection procedures. Therefore, recent studies focus on the improvement of selected strains by the application of recombinant DNA technology. By its application, certain beneficial features, such as health-promoting properties, accelerated acid production, wholesomeness and overproduction of specific enzymes or bacteriocins can be improved (Holzapfel, 2002). Gene dismption may be used to... 

Although chemical techniques can be used to modify the properties of biopolymers in order to expand their range of applications, this is not the unique way to improve biopolymer performance. There are different methods to transform biopolymers in sources of structural polymers that may supplant traditional commodity plastics, such as genetic manipulation of some plant species, polymerization of biological starting materials, or the creation of new gene sequences that can lead to novel protein polymers through the application of recombinant DNA methods. However, only biopolymer physical/chemical modifications will be discussed in this chapter. 

PATENTABILITY. The qualifications for obtaining a patent on an invention of chemical process. These arc (1) the invention must not nave been published in any country or in public use in the U.S., in either case for more than 1 year before the date of filing the application (2) it must not have been known in the IJ.S. before date of invention by the applicant (3) it must not be obvious to an expert in the art (4) it must be useful for a purpose not immoral and not injurious to the public welfare (5) it must fall within the five statutory classes on which patents may be granted, i.e., (a) composition of matter, (b) process of manufacture or treatment, (c) machine, (d) design (ornamental appearance), or (e) a plant produced asexually. Special regulations relate to atomic eneigy developments and subjects directly affecting national security (Robert Calvert). Note In 1980, the Supreme Court m al landmark decision upheld the patentability of synthetic bacteria created by recombinant DNA techniques. 

With these future applications of genetic engineering techniques of plant cells in mind, the recombinant DNA technology for the plant cells is reviewed briefly (Lee and An, 1986). 

The possibility that interferons can be used as pharmacologic agents has aroused a great deal of interest. Recombinant DNA techniques and cell tissue cultures have been used to produce sufficient quantities of interferons for clinical drug trials. The rationale is that exogenously administered interferons will produce antiviral and other beneficial effects in healthy cells in a manner similar to their endogenously produced counterparts. Some of the pertinent aspects of interferon action and clinical applications are presented below. 

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