Biotechnology: Methodology in Basic Genetics

Project Summary

The material presented in this research proposal illustrates the possibilities of ecological genetics, in particular, the development of eco-genetical models, based on the usage of species linked by one food chain as consumers and producers. The consumer’s metabolic dependence on producer allows altering the metabolism of the producer through basic genetic procedures and regulating the development and numbers of the consumer, influencing its genetic processes, which are decisive in terms of population dynamics. Besides, such approach is prospective for plant selection and may be an alternative to elimination of harmful insects by pesticides.

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Sterines are vital compounds required by insects and are synthesized in plants. Therefore, most parasitic insects obtain sterines from exogenous sources (Stanley-Samuelson and Nelson 1993). This project focuses on altering metabolism of sterines in plant cells in order for them to become uneatable by pests. The model of yeast-drosophila and plant-drosophila is used to demonstrate the important role of sterines in the insect metabolism.

Tobacco, peas, and potatoes are not affected by drosophila, and selection of plant cells in this project shows nothing more than laboratory model research that is far from real agricultural practices. Although this is a model study, the biological laws that are its basis are universal, and through these methods it is possible to conduct selection, joining any agricultural plant with an insect that harms it.

Furthermore, let me remind you that the requirement of exogenous source of sterines is a known feature of not solely insects, but also nematodes, and some parasitic fungi such as potato blight (Phytophthora infestans), which lost its ability to synthesize the needed b-sitosterol as a result of parasitic specialization (Knobloch 1948). Taking into account this fact, it is possible to predict that mutated potatoes with modified sterine metabolism could be resistant to not just insects, in particular the Corolado beetle, but also to phytophtora that causes severe damage to potato growing.

Introduction

In nature, food chains are patterns of substance and energy flow from one organism to another. At this, the diversity of the nutrition sources of each biological species usually does not allow to present the relations between the predators and their prey in a simple form. Although there are some exceptions, when these interactions can be portrayed as sequential stages of biochemical processes that are proceeding in different types of organisms. One of such examples is being looked at in this research proposal. The metabolism of sterines is vital for insects, as well as for some parasite fungi and nematodes in order to have development.

Insects as a biological class (arthropods) include around one million species. This is more than all the other creatures and plants put together. Thereat they inhabit much more diverse places of habitat and their food is much more broad-ranged comparing to any other type of species. This way, insects are the most prospering biological group on the planet (Morris 2004). Braced by the chitinous armor they are everywhere, and are often infesting agriculture, causing much trouble to the industry. The common peculiarity of insects is the hard external skeleton lays certain distinctions on their development. As the specimen grows, its hard shell becomes small, and the insect has to get rid of it. The insects are molting a few times during the course of life.

The molting hormones are ecdysones. They are related to steroid hormones and appear to be derivatives of cyclopentanperhydrophenanthrene, for example a-ecdisone (Scharrer, and Scharrer 1963). The insects are unable to synthesize predecessors of ecdysones. For instance, the fruit fly Drosophila melanogaster (the most favorite subject of genetics) obtains a precursor of ecdysones

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— ergosterin, or its closest similar by structure cholesterol via nutrition (Dobzhansky, and Epling 1984). The source of these compounds appears to be plain baker’s yeast Saccharomyces cerevisiae, which actively develops on rotting fruits and vegetables (Burdette 1963). Yeasts are purposely included into the diet of Drosophila, as they represent an essential component of its laboratory nutritional medium.

In natural conditions the sources of essential for insects sterines are plants, mushrooms, animals, and other organisms, which are autonomous in this sense, hence they are able to synthesize irreplaceable sterines included into their cellular membranes and acting as precursors of steroid hormones (Wynne-Edwards 2001). In this way, the prosperity of the insects is related to the surrounding organisms, as well conditioned by dependable functioning of sequential stages of the food chain called producer – consumer of sterines.

Experimental approach

The above condition was a good reason for developing an elementary ecological-genetic model called yeast-drosophila. Both subjects are well studied genetically wise. The genetic control of concluding stages of sterine biosynthesis in yeast is well known due to the fact that it can be blocked by mutations of resistance to a polyene antibiotic nystatin, and other antibiotics from the same group, which are usually bonding with the sterines of the cellular membranes and prevent cell multiplying (Menditto, and Kirsch 1983). The wild type yeasts do not grow on the medium with nystatin.

If the mutation is blocking the synthesis of ergosterin, the yeasts become resistant to the antibiotic (Nysr) and grow on the same medium there is a simple selection method for obtaining yeast mutants with altered metabolism of sterines.

It turned out that adding some mutant types of Nysr, in particular Nysr 1 (erg 6), as the one and only source of sterines into the cultural medium for the drosophila blocks the development of the insect. If the flies are laying eggs onto such medium, the hatched larvae dies, and if the adult females are feeding in such medium, they become sterile and cannot lay eggs even in normal medium with wild type yeast, where the female drosophila is usually kept, or lays defective eggs that are unable to develop. Other yeast mutants have led to less expressive effect. The reason for the obtained anomaly is the deficit of sterines in the diet of drosophila.

This can be easily checked. If ergosterin or cholesterol is added to the food that contains mutated yeasts Nysr1, the development and fertility of drosophila is being restored. At this, if the sterines are added in concentrations lower than the optimal (0.01%), which influences the ecological relation of the two species, the development of fertility of drosophilae can be adjusted according to the wishes of experimenter.

The above model has allowed us to pose a question: does the modification of ecological relations reflect on the intraspecific variation of the consumer species that is an important factor of micro evolutionary processes? It turned out that in under the conditions of sterine deficit, when the flies were feeding on mutated yeasts Nysr1, their crossing-over frequency – rate of recombination of homological chromosomes had significantly reduced, whereas the rate of chromosomal x-ray induced losses had significantly increased. At this, just as in case of the study of drosophila breeding performance, adding of cholesterol to the diet normalizes the situation. The rate of chromosomal losses decreases to the level which is common for flies feeding on normal non-mutated yeasts.

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In this fashion, the hereditary variance is a powerful factor of the evolutionary process, and it depends on the type of ecological relations between species, as well as these relations can be modeled under the laboratory experiment conditions due to identification and exposure of an elementary unit character – certain stage in the sterine biosynthesis that provides such relations. These experiments are of great interest in terms of developing the issues of ecological genetics and further development of the evolution theory.

Let me remind the reader that genetically controlling the metabolism of sterines in the food chain allowed to modify the development and fertility of drosophilae, and consequently regulate the numbers of these consumer species. An important question is whether this same principal can be applied to control the numbers of pests that infest agricultural plants. This idea is not new. The perspective of such approach as an alternative of chemical suppression of the insects and other agricultural pests is evident. It is not worth applying to the methods of total elimination of certain species whose essential interests collide with those of human beings.

First of all, the reduction of biological diversity of our planet is irrational, and in our case impossible according to centuries long experience of out agricultural activity. An attempt to solve the problem head-on using powerful means of chemistry, including agricultural aviation leads to appearance of more and more aggressive forms of pests. Secondly, the detonation of total chemical war with insects, parasitic fungi, and bacteria leads to pollution of the environment by toxic mutagenic and cancerogenic substances. Even the agricultural plants cannot withstand the great doses of chemical pesticides. The offered ways of increasing the plant resistance to these pesticides appear to be at least short-sighted, as there are high chances of pesticides appearing in our food. The problem of human resistance to pesticides is not discussed in this paper.

Considerably more prospective, although somewhat slower method is developed on the basis of the yeast-drosophila model. In order to accomplish this task it is necessary to obtain mutated plants with altered sterine metabolism. In this case the insects will be unable to consume these plants for food. Skeptics often ask a question of whether such plants will be eatable for humans. A preliminary answer for to this question could be illustrated by an example from nature.

A mushroom called Cantharellus cibarius is the only eatable mushroom that does dot get consumed by insects. It appears that Cantharellus cibarius do not contain the type of sterines that the insects require. The only things that are left at this point are technical issues, for example how to obtain and study a large number of mutated plants. This is not as easy as in the case with yeast if we were to rely upon traditional methods of crop raising.

Selection in the Test Tube

The aim is to develop selection methods of plants with altered sterine metabolism. The plant cells shall be cultivated on artificial mediums. Currently it is possible to obtain homogeneous mass of undifferentiated plant cells in a test tube or Petri dish with agar nutrient solution. These cells would not look like the ordinary plant cells, and would rather remind colonies of microorganisms – yeasts or bacteria. These cells are handled in the same manner as microbes, may be obtained in large amounts in order to be subjected to mutagens for inducing mutations, and these mutants can be then picked out on provocative background, for example in mediums with polyene antibiotics or other antimetabolites that disrupt sterine biosynthesis.

If necessary, it is possible to artificially cause differentiation of cultivated cells, by influencing with plant hormones, obtain whole plants, and place them back in the field. It appears that homogenates of plant tissues or cellular mass, which was grown under laboratory conditions is able to replace yeasts in terms of sterine source for drosophila described in the two-species model. In this manner, the yeasts-drosophila model has transformed into plant drosophila model.

Budget Justification

$ 25,000 per year is budgeted towards the individual salary of each experiment assistant.

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$12,000 is required for purchasing the supplies which include plants, mutagens, markers, etc…

$550 per person will enable the experimenters to interact with other state institutions.

$ 2,000 per person will cover travel expenses for attending a conference.

References Cited

Burdette WJ, ed., 1963. Methodology in Basic Genetics (San Francisco): Holden-Day.

Dobzhansky TG, Epling C. 1984. Contributions to the Genetics, Taxonomy, and Ecology of Drosophila Pseudoobscura and Its Relatives. (Washington DC): Carnegie Institution of Washington.

Knobloch IW, ed. 1948. Readings in Biological Science (NY): Appleton-Century-Crofts.

Menditto J, Kirsch D. 1983. Genetic Engineering, DNA, and Cloning A Bibliography in the Future of Genetics. (Troy, NY): Whitston Publishing.

Morris B. 2004. Insects and Human Life. (NY): Berg.

Scharrer E, Scharrer B. 1963. Neuroendocrinology (NY): Columbia University Press.

Stanley-Samuelson DW, Nelson DR, eds. 1993 Insect Lipids: Chemistry, Biochemistry, and Biology (NE): University of Nebraska Press.

Wynne-Edwards KE. 2001. Evolutionary Biology of Plant Defenses against Herbivory and Their Predictive Implications for Endocrine Disruptor Susceptibility in Vertebrates. Environmental Health Perspectives 109(5): 443.

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