Variant Mutant86.11%

Revertant

Revertants

Related terms:

Chinese Hamster Ovary Cell

Salmonella

Phenotypes

Auxotrophic Mutant

Chromatid

Leucocyte

Oxygen Specie

Primary Metabolite

Revertant

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Agricultural and Related Biotechnologies

H. Ashida, A. Yokota, in Comprehensive Biotechnology (Second Edition), 2011

4.13.2.3 Directed Evolution of RuBisCO

Directed evolution is a powerful approach to screen for improved enzymes resulting from random mutations. In directed evolution of RuBisCO, the properties of the newly created enzymes are evaluated by determining the biological performance of the host microorganisam; that is, those showing higher growth rates are expressing an improved RuBisCO. This type of screening method has successfully isolated some improved RuBisCOs. Spreitzer and co-workers identified amino acid residues involved in Kmc and Srel by screening revertants of Chlamydomonas RuBisCO mutants; that is, a successful revertant expressing a remutated RuBisCO with improved enzymatic properties can grow under photoautotrophic conditions [19]. Based on this screening experiment, Spreitzer et al. [17] improved the Chlamydomonas RuBisCO through multiple mutations of V221LC/V235LI/C256LF/K258LR/I265LV and the βA–βB loop replacement of RbcS, as discussed above. Similarly, Tabita and co-workers identified amino acid residues involved in kinetic parameters of cyanobacterial RuBisCO using a screening system with a RuBisCO-deficient mutant of Rhodobacter capsulatus [20].

The directed evolution of RuBisCO has also been carried out using Escherichia coli [21, 22]. E. coli is one of the most suitable hosts for directed evolution of enzymes, because its transformation efficiency is very high (up to 1010 colony-forming units/μg plasmid). In the E. coli system, the gene for glyceraldehyde-3-phosphate dehydrogenase was disrupted and the gene encoding phosphoribulokinase, which catalyzes production of RuBP from ribulose 5-phosphate, was expressed together with the randomly mutated RuBisCO gene. In this screening system, the growth of the hosts relied on the enzymatic properties of RuBisCO to consume RuBP and produce PGA, partly because RuBP is a metabolically dead-end compound and is toxic to E. coli, and partly because the host cannot synthesize PGA. Matsumura and co-workers found that the M259LT mutation improved folding and catalytic efficiency of a cyanobacterial RuBisCO using this E. coli screening system [21]. In the same screening system, Mueller-Cajar and Whitney [22] identified I174LV, K339LR, and N115LS mutations, all of which enhanced Srel in a cyanobacterial RuBisCO (Table 1).

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The isolation and improvement of industrially important microorganisms

Peter F. Stanbury, ... Stephen J. Hall, in Principles of Fermentation Technology (Third Edition), 2017

Isolation of mutants that do not recognize the presence of inhibitors and repressors

The use of auxotrophic mutants has resulted in the production of many microbial products in large concentrations, but, obviously, such mutants are not suitable for the synthesis of products that control their own synthesis independently. A hypothetical example is shown in Fig. 3.21 where the end product P controls its own biosynthesis by feedback inhibition of the first enzyme in the pathway. If it is required to produce the intermediate F in large concentrations then this may be achieved by the isolation of a mutant auxotrophic for P, blocked between F and P. However, if P is required to be synthesized in large concentrations it is quite useless to produce an auxotrophic mutant. The solution to this problem is to modify the organism such that the first enzyme in the pathway no longer recognizes the presence of inhibiting levels of P. The isolation of mutants altered in the recognition of control factors has been achieved principally by the use of two techniques:

Figure 3.21. The Control of the Production of an End Product P

The isolation of analog resistant mutants.

The isolation of revertants.

An analog is a compound that is very similar in structure to another compound. Analogs of amino acids and nucleotides are frequently growth inhibitory, and their inhibitory properties may be due to a number of possible mechanisms. For example, the analog may be used in the biosynthesis of macromolecules resulting in the production of defective cellular components. In some circumstances the analog is not incorporated in place of the natural product but interferes with its biosynthesis by mimicking its control properties. For example, consider the pathway illustrated in Fig. 3.21 where the end product, P, feedback inhibits the first enzyme in the pathway. If P* were an analog of P (which could not substitute for P in biosynthesis) and were to inhibit the first enzyme in a similar way to P, then the biosynthesis of P may be prevented by P* which could result in the inhibition of the growth of the organism.

Mutants may be isolated which are resistant to the inhibitory effects of the analog and, if the site of toxicity of the analog is the mimicking of the control properties of the natural product, such mutants may overproduce the compound to which the analog is analogous. To return to the example of the biosynthesis of P where P* is inhibitory due to its mimicking the control properties of P; a mutant may be isolated which may be capable of growing in the presence of P* due to the fact that the first enzyme in the pathway is no longer susceptible to inhibition by the analog. The modified enzyme of the resistant mutant may not only be resistant to inhibition by the analog but may also be resistant to the control effects of the natural end product, P, resulting in the uninhibited production of P. If the control system were the repression of enzyme synthesis, then the resistant mutant may be modified such that the enzyme synthesis machinery does not recognize the presence of the analog. However, the site of resistance of the mutant may not be due to a modification of the control system; for example, the mutant may be capable of degrading the analog, in which case the mutant would not be expected to overproduce the end product. Thus, analog resistant mutants may be expected to overproduce the end product to which the analog is analogous provided that:

The toxicity of the analog is due to its mimicking the control properties of the natural product.

The site of resistance of the resistant mutant is the site of control by the end product.

Resistant mutants may be isolated by exposing the survivors of a mutation treatment to a suitable concentration of the analog in growth medium and purifying any colonies that develop. Sermonti (1969) described a method to determine the suitable concentration. The organism was exposed to a range of concentrations of the toxic analog by inoculating each of a number of agar plates containing increasing levels of the analog with 106–109 cells. The plates were incubated for several days and examined to determine the lowest concentration of analog which allowed only a very few isolated colonies to grow, or completely inhibited growth. The survivors of a mutation treatment may then be challenged with the predetermined concentration of the analog on solid medium. Colonies that develop in the presence of the analog may be resistant mutants.

Szybalski (1952) constructed a method of exposing the survivors of a mutation to a range of analog concentrations on a single plate. Known as the gradient plate technique, it consists of pouring 20 cm3 of molten agar medium, containing the analog, into a slightly slanted petri dish and allowing the agar to set at an angle. After the agar has set, a layer of medium not containing the analog is added and allowed to set with the plate level. The analog will diffuse into the upper layer giving a concentration gradient across the plate and the survivors of a mutation treatment may be spread over the surface of the plate and incubated. Resistant mutants should be detected as isolated colonies appearing beyond a zone of confluent growth, as indicated in Fig. 3.22. Whichever method is used for the isolation of analog-resistant mutants, great care should be taken to ensure that the isolates are genuinely resistant to the analog by streaking them, together with analog-sensitive controls, on both analog-supplemented and analog-free media. The resistant isolates should then be screened for the production of the desired compound by over layering them with a bacterial strain requiring the compound; producers may then be recognized by a halo of growth of the indicator strain.

Figure 3.22. The Gradient Plate Technique for the Isolation of Analog-Resistant Mutants

Sano and Shiio (1970) investigated the use of lysine analog-resistant mutants of Brevibacterium flavum for the production of lysine. The control of the biosynthesis of the aspartate family of amino acids in B. flavum is as illustrated for C. glutamicum in Fig. 3.16. The main control of lysine synthesis is the concerted feedback inhibition of aspartokinase by lysine and threonine. Sano and Shiio demonstrated that S-(2 aminoethyl) cysteine (AEC) completely inhibited the growth of B. flavum in the presence of threonine, but only partially in its absence. Also, the inhibition by AEC and threonine could be reversed by the addition of lysine. This evidence suggested that the inhibitory effect of AEC was due to its mimicking lysine in the concerted inhibition of aspartokinase. AEC-threonine-resistant mutants were isolated by plating the survivors of a mutation treatment on minimal agar containing 1 mg cm–3 of both AEC and threonine. A relatively large number of the resistant isolates accumulated lysine, the best producers synthesizing more than 30 g dm–3. Investigation of the lysine producers indicated that their aspartokinases had been desensitized to the concerted inhibition by lysine and threonine.

The development of an arginine-producing strain of B. flavum by Kubota, Onda, Kamijo, Yoshinaga, and Oka-mura (1973) provides an excellent example of the selection of a series of mutants resistant to increasing levels of an analog. The control of the biosynthesis of arginine in B. flavum is similar to that shown for C. glutamicum in Fig. 3.17. Kubota et al. selected mutants resistant to the arginine analog, 2-thiazolealanine, and the genealogy of the mutants is shown in Fig. 3.23. Strain number 352 produced 25.3 g dm–3 arginine. Presumably, the mutants were altered in the susceptibility of the second enzyme in the pathway to inhibition by arginine.

Figure 3.23. The Genealogy of l-Arginine-Producing Mutants of B. flavum

TA, Thiazolealanine; NG, N-methyl-N´-nitro-N-nitroso-guanidine (Kubota et al., 1973).

The second technique used for the isolation of mutants altered in the recognition of control factors is the isolation of revertant mutants. Auxotrophic mutants may revert to the phenotype of the mutant "parent." Consider the hypothetical pathway illustrated in Fig. 3.21 where P controls its own production by feedback inhibiting the first enzyme (a) of the pathway. A mutant does not produce the enzyme, a, and is, therefore, auxotrophic for P. However, a revertant of the mutant produces large concentrations of P. The explanation of the behavior of the revertant is that, with two mutations having occurred at loci concerned with the production of enzyme a, the enzyme of the revertant is different from the enzyme of the original prototrophic strain and is not susceptible to the control by P. Revertants may occur spontaneously or mutagenic agents may be used to increase the frequency of occurrence, but the recognition of the revertants would be achieved by plating millions of cells on medium which would allow the growth of only the revertants, that is, in the earlier example, on medium lacking P.

Shiio and Sano, 1969 investigated the use of prototrophic revertants of B. flavum for the production of lysine. These workers isolated prototrophic revertants from a homoserine dehydrogenase-defective mutant. The revertants were obtained as small-colony forming strains and produced up to 23 g dm–3 lysine. The overproduction of lysine was shown to be due to the very low level of homoserine dehydrogenase in the revertants that presumably, resulted in the synthesis of threonine and methionine in quantities sufficient for some growth, but insufficient to cause inhibition or repression.

Mutant isolation programs for the improvement of strains producing primary metabolites did not rely on the use of only one selection technique. Most projects employed a number of methods including the selection of natural variants and the selection of induced mutants by a variety of means. The selection of bacteria overproducing threonine provides a good example of the use of a variety of selection techniques. Attempts to isolate auxotrophic mutants of C. glutamicum producing threonine were unsuccessful despite the fact that productive auxotrophic strains of Escherichia coli had been isolated. Huang (1961) demonstrated threonine production at a level of 2–4 g dm–3 by a diaminopimelate and methionine double auxotroph of E. coli. Kase, Tanaka, and Nakayama (1971) isolated a triple auxotrophic mutant of E. coli that required diaminopimelate, methionine, and isoleucine and produced between 15 and 20 g dm–3 threonine. The control of the production of the aspartate family of amino acids in E. coli is shown in Fig. 3.24 and that in C. glutamicum in Fig. 3.16. The mechanism of control in E. coli involves a system of isoenzymes, three isoenzymic forms of aspartokinase and two of homoserine dehydrogenase, under the influence of different end products. However, in C. glutamicum control is effected by the concerted inhibition of a single aspartokinase by threonine and lysine; by the inhibition of homoserine dehydrogenase by threonine and the repression of homoserine dehydrogenase by methionine. Thus, the control of homoserine dehydrogenase may not be removed by auxotrophy without the loss of threonine production. However, in E. coli methionine auxotrophy would remove control of the methionine-sensitive homoserine dehydrogenase and aspartokinase that would still allow threonine production, despite the control of the threonine-sensitive isoenzymes by threonine. E. coli mutants also lacking lysine and isoleucine would be relieved of the control of the lysine-sensitive aspartokinase and the degradation of threonine to isoleucine.

Figure 3.24. Control of the Aspartate Family of Amino Acids in E. coli

The production of threonine by C. glutamicum was achieved by the use of combined auxotrophic and analog resistant mutants. A good example of the approach is given by Kase and Nakayama (1972) who obtained stepwise improvements in productivity by the imposition of resistance to α-amino-β-hydroxyvaleric acid (a threonine analog) and S-(β-aminoethyl)-l-cysteine (a lysine analog) on a methionine auxotroph of C. glutamicum. The genealogy of the mutants is shown in Fig. 3.25. The analog-resistant strains were altered in the susceptibility of aspartokinase and homoserine dehydrogenase to control, and the lack of methionine removed the repression control of homoserine dehydrogenase. The use of transduction and recombinant DNA technology has resulted in the construction of far more effective threonine producers and these strains are considered in later sections of this chapter.

Figure 3.25. The Genealogy of Mutants of C. glutamicum Producing l-Threonine or l-Threonine Plus l-Lysine

AHV, α-Amino-β-hydroxy valeric acid; AEC, S-(β-aminoethy)-l-cysteine (Kase & Nakayama, 1972).

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Carotenoids and Cancer: Basic Research Studies

Norman I. Krinsky, in Natural Antioxidants in Human Health and Disease, 1994

B Effects on Genotoxicity

Genotoxicity is not a very specific term, but can cover a variety of insults to the genome, which can be expressed as DNA damage, formation of micronucleated cells, sister chromatid exchanges (SCEs), chromosomal aberrations, translocations, or even death of the cell. In the following reports, these various aspects of genotoxicity have been modified by the addition of carotenoids.

Polymorphonuclear leukocytes (PMNs) not only play an important defensive role elaborating reactive oxygen species to help destroy invading organisms, but can also be harmful to tissues when there is an overproduction of these same reactive oxygen species (see also Chapters 2 and 15, this volume). Tumor promoters, such as phorbol myristate acetate, have a powerful effect in eliciting release of reactive oxygen species from PMNs. Weitzman and Stossel (1981) have studied the effects of adding activated PMNs to S. typhimurium (TA 102), and observed an increased number of histidine revertants, indicative of the occurrence of mutagenesis. In addition to inducing mutagenesis, activated PMNs can cause an increase in the frequency of SCE when they are placed on cultures of Chinese hamster ovary (CHO) cells (Weitzman and Stossel, 1982). Weitzman et al. (1985) used either phorbol myristate acetate-stimulated PMNs, or xanthine oxidase and hypoxanthine, to generate reactive oxygen species directly to induce increased SCE in CHO cells. When 10–50 μM β-carotene was added to the cell culture system, a significant protection against the generation of SCE was observed. However, the significance of these findings may not be extended to in vivo situations. The total concentration of carotenoids in human serum is in the 1.5–3 μM range, with β-carotene representing about 30% (Krinsky et al., 1990), so the concentrations used above should be considered high by human physiological standards. The group of Anderson has determined the extent of SCE in PMNs from smokers, and did not find a significant correlation with serum concentrations of either ascorbate, β-carotene, or α-tocopherol (van Rensburg et al., 1989).

When either methylmethane sulfonate or 4-nitroquinoline-1-oxide is added to CHO cells growing in culture, a variety of genotoxic symptoms appear, including chromosomal aberrations, translocations, or the development of micronuclei (Stich and Dunn, 1986). The addition of β-carotene to these cultures resulted in a dose-dependent inhibition of genotoxicity, and because the authors were not able to detect retinol in these cells, they concluded that the inhibitory effects of β-carotene were not associated with its conversion to retinol. The CHO cells were not protected by β-carotene against other genotoxic compounds such as gallic acid, tannic acid, aqueous extract of the areca nut, or hydrogen peroxide. In a related study, Stich et al. (1990) measured the chromosome instability of C127 cells transformed with bovine papillomavirus. A number of antioxidants, including β-carotene, canthaxanthin, retinoic acid, retinol, ascorbate, and ellagic acid, when added to the cultures at physiological concentrations (β-carotene and canthaxanthin at 0.5 μM), were able to decrease the papillomavirus-induced chromosome instability.

Banerjee and associates (Manoharan and Banerjee, 1985) have used mouse mammary cell organ cultures treated with chemical carcinogens such as dimethyl-benzanthracene (DMBA) to induce SCE. When β-carotene was added to the medium during the 24-hr initiation stage, there was a significant decrease in the number of SCEs. They also noted a similar protective effect when two other carcinogens, N-nitrosodiethylamine and methylnitrosourea, were studied.

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YERSINIA | Yersinia enterocolitica

S. Bhaduri, in Encyclopedia of Food Microbiology (Second Edition), 2014

Isolation of Pathogenic YEP+ Strains from Foods

Common food vehicles in outbreaks of yersiniosis are meat (particularly, pork), milk, dairy products, powdered milk, cheese, tofu, and raw vegetables. Most strains isolated from these foods differ in biochemical and serological characteristics from typical clinical strains and are usually called nonpathogenic or environmental Yersinia strains. The increasing incidence of Y. enterocolitica infections and the role of foods in some outbreaks of yersiniosis have led to the development of a wide variety of methods for the isolation of YEP+ strains from foods. Since the population of YEP+ strains in food samples is usually low and the natural microflora tend to suppress the growth of this organism, isolation methods usually involve enrichment followed by plating on selective media. Food matrices can also inhibit the enrichment of YEP+ strains.

The efficiency of pYV-bearing Y. enterocolitica enrichment techniques varies with serotype and depends on the type of food being tested. Different enrichment procedures have been described to recover the full range of YEP+ serotypes from a variety of foods. The unstable nature of the virulence plasmid complicates the isolation of pYV-bearing virulent Y. enterocolitica by causing the overgrowth of virulent cells by plasmidless revertants, eventually leading to a completely avirulent culture. Traditional methods employ prolonged enrichment at refrigeration temperatures to take advantage of the psychrotrophic nature of Y. enterocolitica and to suppress the growth of background flora. Due to the extended time period needed for this method, efforts have been made to devise selective enrichment techniques employing shorter incubation times and higher temperature, making them more practical for routine use. High levels of indigenous microorganisms can overgrow and mask the presence of YEP+ strains, including nonpathogenic Y. enterocolitica strains. Enrichment media containing selective agents such as Irgasan, ticarcillin, and potassium chlorate are effective in recovering a wide spectrum of YEP+ strains from meat samples. No single enrichment procedure, however, has been shown to recover a broad spectrum of pathogenic Y. enterocolitica.

Since there is no specific plating medium for the isolation of YEP+ strains, cefsulodin–irgasan–novobiocin (CIN) and MacConkey agars are commonly used to isolate presumptive Y. enterocolitica from foods. The initial isolation of presumptive Y. enterocolitica from enriched samples on CIN and MacConkey agars adds an extra plating step, and picking presumptive Y. enterocolitica requires skilled recognition and handling of the colonies. The unstable nature of the virulence plasmid complicates the detection of YEP+ strains, since isolation steps may lead to plasmid loss and the loss of associated phenotypic characteristics for colony differentiation. Since colonies of Y. enterocolitica are presumptive on the plating media, these isolates should be verified as YEP+ strains. Biochemical reactions, serotyping, biotyping, and virulence testing are essential for differentiation between YEP+, YEP−, environmental Yersinia strains, and other Yersinia-like presumptive organisms. Biochemical tests using systems such as API 20E give similar reactions among these organisms and are not conclusive. Serotyping involving major O and H factors differentiate between pathogenic and environmental Y. enterocolitica but fail to discriminate between YEP+ and YEP− strains. Biotyping involves biochemical tests, which do not detect the presence of the pYV. Thus, it does not identify YEP+ strains. Several pYV-associated phenotypic virulence characteristics, including colony morphology, AA, serum resistance, tissue culture detachment, HP, Lcr, CV-binding, CR-binding, isolation of pYV, and colony hybridization techniques have been described to determine the potential virulence of Yersinia isolates. Unfortunately, methods described in the literature do not treat confirmation of virulence in presumptive or known Y. enterocolitica isolates recovered from selective agars as an integral part of the detection method. The fastest enrichment procedure available for the isolation of a wide spectrum of Y. enterocolitica strains does not include the verification of isolates as YEP+ strains.

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GMO Acceptance in the World and Issues for the Overcoming of Restrictions

P. Poltronieri, in Biotransformation of Agricultural Waste and By-Products, 2016

12.2 Non-GMO Methods for Plants Improvement: Mutagenesis

Plant products produced by conventional breeding are more familiar to consumers. In the last twenty years, plants with mutated genes have been introduced in agriculture, since their production confers the ability to eliminate weeds or to grow them in adverse conditions (excessive salinity, adverse climate conditions). Many gene-editing events have been the result of random mutagenesis.

The induction of mutations using various types of mutagenic agents has been used worldwide for crops and trees. At the beginning of 2000, the number of mutated varieties was around 2252 (FAO, 2000), including varieties obtained by crosses with mutated varieties, with cereals summing up to 1172 cases, making the largest part of the total number.

In Italy, 35 varieties obtained by induced mutagenesis have been released to market, among them, 13 varieties of wheat (Triticum turgidum sbsp. durum) (Creso variety), 6 for pea, 2 for T. aestivum, 3 for eggplant, 1 for olive tree, 1 potato, and 1 for rice (Fulgente variety in 1973, obtained from Maratelli by X-ray mutagenesis).

According to the United Nations Food and Agricultural Organization (FAO), a great number of rice mutated varieties, 434 at the beginning of this century, were present in Italy, Asia, US, Australia, Egypt, and South America.

Considering that the new characters have been obtained through selection and genetic improvement starting from mutant varieties crossed with well-performing ones, the general opinion is that these plants are not transgenic, since any gene, or DNA sequence, from other genomes has been introduced but has passed through an accelerated evolution process.

Therefore, it is at the attention of authorities and regulatory framework that when plants have maintained genome integrity and only one nucleotide substitution is cause of the gene-based phenotype, these plants are assimilated to those obtained by advanced breeding methods. The same equivalence should be granted, or the methods will be assimilated to accelerated evolution and enhanced breeding-like processes when methods for precise editing of genomes, such as CRISPR-cas system, TALENs, and ZFNs will provide more precise intervention protocols without any other change in plant genomes.

12.2.1 Insertional Mutagenesis

An important and direct approach to defining the function of a novel gene is to abolish or activate its function by mutagenesis with physical or chemical mutagens and modern tools such as targeting-induced local lesions in genomes (TILLING) and NGS.

Transposons are genetic mobile elements that occasionally move from one DNA position to another, causing the inactivation of genes, and are based on a transposase and long terminal repeat (LTR) sequences. Insertional mutagenesis, with T-DNA (the transfer DNA on Agrobacterium plasmids) or a transposable element, provides opportunities for assigning a function to a particular DNA sequence and isolating the target gene, causing a specific phenotype.

Ds and Spm maize transposable elements have been introduced in other crops to create lines carrying new transposon inserts. The maize Ac/Ds transposon system has been used to generate an insertional mutant population in maize itself, Arabidopsis, and rice. An Ac/Ds-based library has several advantages: (1) revertants can be readily obtained and easily identified; and (2) Ds elements prefer to transpose to genetically linked sites (i.e. the same chromosome).

Ac/Ds belong to the hAT super family, with the designation hAT from the Drosophila element hobo, maize element Ac, and Antirrhinum majus Tam3 element. Ac, Ds, and Spm are introduced by Agrobacterium-mediated transformation, and the regenerated rice lines may exhibit somaclonal variation, especially in the first generation, since the variation will tend to be diluted with no further changes in the offspring, once the mutants are crossed with the Ac lines and insert population amplified. New rice hAT elements have been found and are suggested as new candidates to generate insertion mutants in rice. An active 0.6-kb endogenous DNA transposon, nonautonomous DNA-based active rice transposon1 (nDart1), was recently identified to act as a causative fragment. One drawback is the GM nature of the lines obtained using transposons from other species. Though belonging to the same family as Ac/Ds, the use of hAT elements for rice mutagenesis is not subject to the concerns on GM problematics because they are endogenous elements in the rice genome, and their mobilization does not necessitate callus formation. Indeed, the transposition of nDart1 can be triggered by ordinary crossing under natural field conditions. As for other transposons, the remobilization of the element generates a revertant.

12.2.2 Chemical Mutagenesis

Various chemical and physical agents have been applied to induce mutants in plant DNA. Both chemical and ionizing radiation mutagenesis have been routinely used to generate genetic variability in rice varieties.

Among the chemical methods for mutagenesis, ethyl methane sulfonate (EMS) has been widely used in plant studies. In Japan, researchers applied EMS mutagenesis to rice immature embryos immediately after fertilization, and the embryos were allowed to develop to seeds. These seeds were sown to generate M1 plants, in which the majority of EMS mutations were expected to be in the heterozygous state (Fekih et al., 2013; Takagi et al., 2015).

Using this approach, RIKEN leaded researchers have developed a mutant rice variety that is able to grow on salt contaminated fields, possessing a high salt tolerance (Takagi et al., 2015). This will allow reintroduction of rice cultivation in areas touched by the tsunami in the North Kanto area.

MutMap is a whole genome sequencing (WGS)-based method that has been applied to accelerated breeding of a salt-tolerant rice cultivar (Takagi et al., 2015). The gene conferring the improved salt resistance has been identified as OsDSS1 (Tamiru et al., 2015).

Among herbicides used to control weeds, the family of imidazolinones is widely used for the low induction of tolerance, often used after appearance of resistance to glyphosate, metolachlor, or imazethapyr. The imidazolinones family consists of six active ingredients, such as imazamox; each of which controls a different spectrum of weeds. Imidazolinones are active at low dosage on most weeds, have low toxicity to animals, inhibit plant acetolactate synthetase (ALH), also known as acetohydroxy acid synthetase (AHAS), an enzyme required for the production of essential branched chain amino acids such as valine, isoleucine, and leucine. Several crops have been mutagenized with EMS to obtain imidazolinones-tolerant mutants. The technology presently registered by BASF, Clearfield, exploits these pioneering studies by various universities. After being licensed the intellectual property rights, BASF has launched its portfolio of Clearfield mutant crop varieties. The current Clearfield crops (maize, canola, rapeseed, rice, sunflower, wheat, and lentils) have been developed using enhanced plant breeding methods. The Clearfield Production System is one of only a few herbicide-tolerant systems recognized as nontransgenic by international authorities. This provides farmers with an effective agronomic tool along with global market acceptance. The Clearfield Production System uses several different herbicides. Each formulation is custom designed to provide exceptional contact and soil activity for broad spectrum, maximum control of the weed species most likely to plague specific crops in each region, while still offering crop rotation flexibility. Elite seed varieties have been developed to tolerate Clearfield herbicides, so the crops thrive as weeds wither and die.

The Clearfield production system for rice

Clearfield rice seed is a nontransgenic, non-GM crop for rice production devoid of weeds, developed with traditional plant breeding techniques. The rice varieties CL 161 and CFX 18, possessing AHAS with an asparagine substitution at position 653, have been crossed with commercial varieties to produce the hybrid. Libero (Oryza sativa indica) is a hybrid representing 10% of the rice cropped in Italy. In the US, 300,000 ha are cultivated using Clearfield rice, over 25% of rice cultivated surface in the mid-South (Louisiana, Arkansas, Missouri, Mississippi, Texas), and its use has spread also in Brazil, Colombia, Uruguay, Argentina, and Costa Rica. Rice Clearfield hybrids, cultivated following the Clearfield® technology by BASF, are tolerant to Newpath® herbicide, Clearpath™ herbicide, and Beyond® herbicide.

Oryza sativa (L.) var. sylvatica is the main weed contaminating paddy fields. Due to the early and casual seed maturation and dropping into the soil, it is able to scatter around and take the lead at the expense of cultivated rice. To preserve the long-term efficacy of the Clearfield rice technology, certain stewardship practices must be followed, and field evaluation has been assessed (Shan et al., 2007). Clearfield rice producers are asked to help protect and prolong the usefulness of this technology by following specific requirements and recommendations to help prevent weed resistance and gene flow from rice to red rice (Shivrain et al., 2007). When used in a planned sequential program, the Clearfield Production System for rice provides the broadest spectrum control of some of the toughest rice weeds, including red rice, barnyard grass, broadleaf signalgrass, eclipta, hemp sesbania, northern joint-vetch, and many more. Clearfield rice producers are required to help protect and prolong the usefulness of the technology by following specific requirements and recommendations to help prevent weed resistance.

The Clearfield production system for wheat

Winter wheat ranks high in importance as an agricultural crop in the Pacific Northwest states of Washington, Idaho, and Oregon. These states rank 3rd, 7th, and 18th in US winter wheat production with a total estimated value of production of over $633 million in 2000 (National Ag. Statistics Service, 2001). Winter wheat is a winter annual grass that is planted and emerges in the fall, overwinters as a small plant, grows fast and develops tillers in the spring, and is harvested in July and August. Winter annual grass weeds such as jointed goatgrass, downy brome, feral rye, and Italian ryegrass have the same growth cycle as winter wheat and are difficult to control in conventional wheat–fallow rotations. These weeds annually account for millions of dollars of losses in yield and wheat production with reduced quality. There has been moderate success in controlling winter annual grasses in wheat by utilizing multiple-year crop rotations with spring crops and fallow periods, and with chemical control of weeds before and after the wheat crop. Beyond herbicide, the EPA received federal registration for use in Clearfield wheat in December 2001. Spring applications of Beyond can control or suppress summer annual broadleaf weeds, such as common lambsquarters, pigweed, wild buckwheat goatgrass, and feral rye.

12.2.3 Mutagenesis by Physical Agents

Accelerated heavy ion beams, carrying much greater energy than X-rays and gamma rays, have been used in mutation induction (mutagenesis). Researchers at the RIKEN Nishina Center for Accelerator-Based Science, provided with a Cyclotron accelerator, developed protocols to mutate a gene even with only a single particle. The high performance of RIKEN accelerators enables the use of ions of carbon, nitrogen, neon, argon, iron, and other elements, thus offering a greater variation in the types of mutations. Seeds and cuttings from a variety of different plants have been exposed to beams of heavy atomic ions accelerated to half the speed of light, producing mutations and breeding new varieties of flowers, crops, and trees. In contrast to other breeding techniques such as hybridization and gene recombination, the timespan for breeding with heavy ion beams can be shortened to only 2 or 3 years.

Gamma rays. Californian rice Calrose 76 variety has been obtained, treating Calrose rice variety with gamma rays, inducing a single nucleotide mutation in the gene sd1-c (semidwarf). The mutant gene affects the production of the growth hormones gibberellins, thus producing semidwarf plants adapted to submerged cultivation, giving rise to the rice green revolution (Sasaki et al., 2002).

Other mutations have been obtained using this method. Among these, amino acids substitutions at Ala122, Pro197, Ala205, Trp574, Ser653 in ALS gene have been found to induce tolerance to imidazolinones.

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Pyrolysis of Aromatic Heterocyclic Compounds

Serban C. Moldoveanu, in Pyrolysis of Organic Molecules (Second Edition), 2019

General Aspects

A number of heterocyclic aromatic amines (HAAs) are formed by pyrolytic processes, particularly from amino acids in pure form but also from amino acids and other nitrogenous compounds in interaction with sugars and creatinine. Heterocyclic aromatic amines also are generated by pyrolysis from more complex mixtures containing nitrogenous materials like proteins, but without the possibility to determine the precise origin of the HAAs. Because HAAs are mutagenic and suspected carcinogens in humans, and because amino acids and sugars are common in food, the presence of HAAs in food is of particular concern. Some aspects regarding the formation of aromatic heterocyclic amines were discussed in Subchapters 8.5, 13.1, and 16.2. A considerable amount of information on the presence of heterocyclic aromatic amines in food, smoke, and the environment is reported in the literature [1–31]. The information is also summarized in excellent reviews [27,32–35] and in at least one monograph [36].

Several studies were focused on the mutagenic properties of HAAs [32]. These compounds are generated in food when it is cooked at temperatures over 150°C. The range of HAA levels is between 0.1 and 50 ppb, depending on the food and cooking conditions. The HAAs are not only present in cooked red meat, fish, and chicken, but also are present at lower levels in baked and fried foods derived from grain. Mutagenicity of fried beef hamburgers cooked at 230°C was determined to be 800 ± 37 TA98 revertants per gram of cooked material [32] in the Ames/Salmonella test. In the tested fried beef, the reported level of 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) was 3.0 ± 2.0 ng/g, of 2-amino-3,4,8-trimethyl-imidazo[4,5-f]quinoxaline (DiMeIQx) was 1.0 ± 0.18 ng/g, of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) was 0.06 ± 0.03 ng/g, and of 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) was 9.6 ng/g. Heterocyclic aromatic amines were capable of producing both reverse and forward mutations in Salmonella bacteria and forward mutations in Chinese hamster ovary cells (CHO). The number and type of mutations depended on the repair capacity of the cells for both Salmonella and CHO. Also, there were statistically significant increases in the mutations in the pancreas of the "mutamouse" following PhIP exposure.

Based on findings from studies on multiple species of experimental animals, it was shown that heterocyclic amines produced cancer in multiple organs, including the forestomach, cecum, colon, liver, oral cavity, Zymbal gland, mammary gland, and skin. Although evidence from human epidemiology suggests that the consumption of well-done or grilled meat (which may contain HAAs) may be associated with increased cancer risk, the data are insufficient to support the conclusion that this risk is due specifically to certain HAAS present in these foods.

The list of some heterocyclic amines (including two heterocyclic compounds, harmane and norharmane) together with their most common origin is given in Table 16.5.1.

Table 16.5.1. Heterocyclic Amines of Health Concern and Their Most Likely Source Following Pyrolysis

AcronymCompoundLikely Pyrolytic OriginReferenceAαC2-Amino-9H-pyrido[2,3-b]indoleSoybean globulin[1,5,6,16,19,20]4-CH2OH-8-MeIQx2-Amino4-hydroxymethyl-3,8-dimethylimidazo[4,5-f]-quinoxaline[16–18,26]Cre-P-14-Amino-1,6-dimethyl-2-methylamino-1H,6H-pyrrolo[3,4-f]benzimidazol2-5,7-dioneProteins[27]4,8-DiMeIQx2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxalineFried beef[27]7,8-DiMeIQx2-Amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline[27]7,9-DiMeIgQx2-Amino-1,7,9-trimethylimidazo[4,5-f]quinoxaline[27]1,6-DMIP2-Amino-1,6-dimethylimidazo[4,5-b]pyridine[27]Glu-P-12-Amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazoleGlutamic acid[10,13–15,18]Glu-P-22-Aminodipyrido[1,2-α:3′2′-d]imidazoleGlutamic acid[10,13–15]Harmane1-Methyl-9H-pyrido[3,4-b]indoleCoffee[20,23,24]IFP2-Amino-1,6-dimethylfuro[3,2-e]imidazo[4,5-b]pyridine[22,27]IQ2-Amino-3-methylimidazo[4,5-f]quinolineBroiled sardines[10,25]IQx2-Amino-3-methylimidazo[4,5-f]quinoxaline[21,28]Lys-P-13,4-Cyclopentenopyrido[3,2-α]carbazoleLysine[27]MeAαC2-Amino-3-methyl-9H-pyrido[2,3-b]indoleSoybean globulin[1,5,6,10,16]MeIQ2-Amino-3,4-dimethylimidazo[4,5-f]quinolineBroiled sardines[18]8-MeIQx2-Amino-3,8-dimethylimidazo[4,5-f]quinoxalineFried beef[1,18,22,25,26]4-MeIQx2-Amino-3,4-dimethylimidazo[4,5-f]quinoxalineFried beef[1,18,22,25,26]1-Methyl-6-phenylimidazolo[4,5-b]pyridine-2-ylamineAlanine?[27]Norharmane9H-Pyrido[3,4-b]indoleCoffee[13,15,20,23,24]Orn-P-14-Amino-6-methyl-1H-2,5,10,10b-tetraaza-fluorantheneOrnithine[27]4′OH-PhIP2-Amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5-b]pyridine[27]Phe-P-12-Amino-5-phenylpyridinePhenylalanine[2,27]PhIP2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridineSmoke[11,19,22,25]1,5,6-TMIP2-Amino-1,5,6-trimethylimidazo[4,5-b]pyridine[27]Trp-P-13-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indoleTryptophan[1,2,5–7]Trp-P-23-Amino-1-methyl-5H-pyrido[4,3-b]indoleTryptophan[2,5–7,10]TriMeIQx2-Amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline[27]

Note: Uncertain origin of pyrolysis product.

The list of detected HAAs is not limited to those listed in Table 16.5.1. Other similar compounds, such as tetrahydro-β-carbolines [37], were reported in specific samples.

Studies on HAAs also were directed toward the measuring of certain markers of exposure to these compounds [38]. For example, after ingestion by different animals, only a few percent of the initial levels of MeIQx and PhIP were excreted as parent compounds. It was determined that urinary levels of parent HAA reflect only recent exposure. The excreted glucuronide conjugates of N-hydroxy-PhIP and N-hydroxy-MeIQx could be markers for the N-hydroxylation capacity and HAA exposure. 5-OH-PhIP can be considered a metabolite marker for PhIP because it is formed from this compound as a by-product along with the formation of PhIP-DNA adducts. PhIP also gives adducts of serum albumin and hemoglobin. In mice, PhIP is irreversibly incorporated in a dose-dependent manner into hair. In humans exposed to an ordinary diet, it was found that the level of PhIP can vary from < 50 pg to 5000 pg PhIP/g hair. It was suggested that this measurement could provide an indication of the level of exposure to PhIP [38].

Other studies attempted to find the metabolic path of HAAs [28,39]. It is likely that the first step in metabolic activation of mutagenic and carcinogenic heterocyclic amines is an N-hydroxylation by cytochrome P-448. N-Hydroxyamino compounds are further activated to form N-O-acyl derivatives that readily react with DNA. The adducts between the metabolites of Trp-P-2 and Glu-P-1 and DNA were shown to have a C8-guanylamino structure [39]. In the case of Glu-P-1, the modification of guanine in GC clusters occurs preferentially. It was shown also that glutathione transferases and myeloperoxidase inactivated some heterocyclic amines or their active metabolites [39]. Also, hemin and fatty acids bind to HAAs and inactivate them. Fibers and other factors from vegetables also work to inactivate heterocyclic amines. Nitrite at low pH degraded some heterocyclic amines, but those with an imidazole moiety were found to be resistant. Glu-P-1 induced intestinal tumors in a high incidence when fed orally to rats. When 14C-Glu-P-1 was administered by gavage into rats, about 50% and 35% were excreted into feces and urine, respectively, within 24 h. When the bile was collected, around 60% of radioactivity was found excreted into it within 24 h. In the bile, N-acetyl-Glu-P-1 was identified as one of the metabolites of Glu-P-1 [39].

In a different study [40], the metabolic activation of IQ to mutagenic intermediates in the Ames test was studied with hepatic activation systems from control and IQ-treated rats. Hepatic S9 preparations from IQ-treated rats were more efficient than the control in converting IQ to mutagens. An increase was seen also when isolated microsomes were employed as activation systems, but this was less pronounced. The microsome-mediated mutagenicity of IQ was potentiated by the addition of the cytosolic fraction from control and IQ-treated rats, with the latter being more effective. It was concluded that IQ, at the doses employed in the study, enhances its own bioactivation to genotoxic metabolites by stimulating both its microsomal and cytosolic metabolisms.

Several theoretical studies were performed with the aim to correlate physicochemical characteristics of HAAs with their carcinogenic properties [41,42]. In one of these studies [42], 11 possible 2-amino-trimethylimidazopyridine isomers were tested for mutagenic potency in the Ames/Salmonella test with bacterial strain TA98. These compounds are related to those found in heated muscle meats. Structural, quantum chemical, and hydropathic data were calculated on the parent molecules and the corresponding nitrenium ions. The principal determinants of higher mutagenic potency in these amines were indicated to be (1) a small dipole moment, (2) the combination of a b-face ring fusion and an N3-methyl group, (3) a lower calculated energy of the π electron system, (4) a smaller energy gap between the amine HOMO and LUMO orbitals (Pearson "softness") (HOMO = highest occupied molecular orbital, LUMO = lowest unoccupied molecular orbital), and (5) a more stable nitrenium ion.

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Revertant mosaicism by somatic reversion of inherited mutations has been described for a number of genetic diseases. Several mechanisms can underlie this reversion process, such as gene conversion, crossing-over, true back mutation, and second-site mutation. Here, we report the occurrence of multiple corrections in two unrelated probands with revertant mosaicism of non-Herlitz junctional epidermolysis bullosa, an autosomal recessive genodermatosis due to mutations in the COL17A1 gene. Immunofluorescence microscopy and laser dissection microscopy, followed by DNA and RNA analysis, were performed on skin biopsy specimens. In patient 1, a true back mutation, 3781T→C, was identified in the specimen from the arm, and a second-site mutation, 4463-1G→A, which compensated for the frameshift caused by the inherited 4424-5insC mutation, was identified in the 3′ splice site of exon 55 in a specimen from the middle finger. Patient 2 showed—besides two distinct gene conversion events in specimens from the arm and hand sites, both of which corrected the 1706delA mutation—a second-site mutation (3782G→C) in an ankle specimen, which prevented the premature ending of the protein by the 3781C→T nonsense mutation (R1226X). Thus, both inherited mutations, paternal as well as maternal, reverted at least once by different reversion events in distinct cell clusters in the described patients. The occurrence of multiple correcting mutations within the same patient indicates that in vivo reversion is less unusual than was generally thought. Furthermore, in the male patient, mosaic patterns of type XVII collagen–positive keratinocytes were present in clinically unaffected and affected skin. This latter observation makes it likely that reversion may be overlooked and may happen more often than expected.We isolated revertant and resistant clones from multidrug-resistant K562/ADM cells and evaluated the expression of P-glycoprotein and the DNA copy number of MDR1. The 9 revertant clones contained 2- to 26-fold DNA copies of MDR1; however, they expressed an extensively decreased P-glycoprotein compared with K562/ADM, while the 10 multidrug-resistant clones contained 4- to 48-fold DNA copies, and the expression level of P-glycoprotein was dependent on the copy number of MDR1 DNA. The decreased expression of P-glycoprotein in the revertants was not due only to the loss of the copy number of MDR1 DNA.

To elucidate the mechanism of P-glycoprotein expression decrease in the revertants, a revertant clone (R1-5) was fused with a multidrug-resistant clone (A2-1) or with a drug-sensitive clone isolated from K562. Compared with K562 clone, the A2-1 contained 32-fold MDR1 DNA copies and showed 131-fold resistance to Adriamycin. The revertant clone R1-5 contained 26-fold MDR1 DNA copies but expressed only 5% the P-glycoprotein of A2-1 cells and showed only 2-fold resistance to Adriamycin. For selection of intraspecific hybrids, a neomycin-resistant or a blasticidin S-resistant gene was introduced into clones by electroporation of pSV2neo or pSV2bsr. The introduction of these resistant genes did not alter the copy number or expression of MDR1 in the clones. Hybrid cells between R1-5bsr and A2-1neo were found to express 136 ± 15% of the P-glycoprotein of A2-1 cells evaluated by quantitative flow cytometry. These hybrid cells contained 41- to 48-fold MDR1 copies and showed the multidrug-resistant phenotype, such as decrease of rhodamine 123 accumulation and 120- to 210-fold resistance to Adriamycin (compared with K562), indicating that the 'silent' MDR1 genes in the revertant clone R1-5 were activated by cell fusion with an MDR clone. R1-5bsr × K562neo hybrids were found to contain 8- to 11-fold MDR1 copies and there was no increase in P-glycoprotein expression as compared with R1-5.Neuromuscular Disorders

Volume 20, Issue 5, May 2010, Pages 295-301

Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: Implication for clinical trials

Author links open overlay panelVirginiaArechavala-GomezaaFrancescoMuntonia

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https://doi.org/10.1016/j.nmd.2010.03.007Get rights and content

Abstract

Duchenne muscular dystrophy (DMD) is characterised by the absence of dystrophin in muscle biopsies, although residual dystrophin can be present, either as dystrophin-positive (revertant) fibres or traces. As restoration of dystrophin expression is the end point of clinical trials, such residual dystrophin is a key factor in recruitment of patients and may also confound the analysis of dystrophin restoration in treated patients, if, as previously observed in the mdx mouse, revertant fibres increase with age. In 62% of the diagnostic biopsies reports of 65 DMD patients studied, traces or revertants were recorded with no correlation between traces or revertants, the patients' performance, or corticosteroids response. In nine of these patients, there was no increase in traces or revertants in biopsies taken a mean of 8.23 years (5.8–10.4 years) after the original diagnostic biopsy. This information should help in the design and execution of clinical trials focused on dystrophin restoration strategies.

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