The world of GMO: from discovery to analysis (part 1)

A GMO (genetically modified organism) is by definition an organism in which the DNA has been modified in a different way from what occurs in nature.

In fact, the production of a GMO is attributed to the hand of man, who using biotechnologies brings changes to the genome. In this case, what takes place is the transfer of genes from one organism to another, even between unrelated species.

Beyond all, the development and use of genetic modification allows man to create organisms such as plants, bacteria, and seeds with characteristics that make them more adapted to survival in nature, with subsequent additional nutritional benefits for humans.

The story that led to the creation of the first GMO products is made up of three elements, and bacteria are involved in all of them.

GMO and Bacillus thuringiensis

Historically, to preserve corn plantations from the attack of the borer (Fig. 1), a peculiar larva that destroys corn kernels and other types of vegetables, these plants were treated with chemical insecticides.

Larva di piralide
Figure 1 – Borer larva [credits:Β informatoreagrario]

However, it was noted that, in some cases, plantations not treated with chemical insecticides were not subject to the attack of the aforementioned larva. Such plantations were called self-protected.

Studying this phenomenon, it was noticed that on the surface of the leaves of these plantations the presence of Bacillus thuringiensis (Fig. 2) was recurrent. This is a Gram + bacterium that carries out sporulation, whose characteristic is to possess, in addition to the mother cell and the prespora inside it, a so-called parasporal body.

Bacillus Thuringiensis
Figure 2 – B. Thuringiensis [credits:Β slideplayer]

In particular, this structure contains a crystalline protein, so abundant that it precipitates and crystallizes within the bacterium’s cytoplasm. For this reason, it was called “cry” protein.

Cry protein

By isolating this protein from B. Thuringiensis and administering it to borer larvae, it was observed that it was solubilized within the gastric apparatus of the animal.

protein Cry GMO
Figure 3 – Cry structure [credits: wikipedia]

Specifically, solubilizing, the protein undergoes an activation process and binds particular surface receptors of the cells of the middle intestine of the larva.

In this way, pores are generated inside the intestinal cells of the larva, causing their death by lysis.

Furthermore, through these pores, bacteria that may be ingested or that are residing in the intestine of the borer are able to cross the barrier of intestinal cells and reach the bloodstream, generating septicemia and the death of the larva.

On the other hand, in the human being, the cry protein is a protein like many others: it is digested in the stomach by proteases and continues to be further degraded in the duodenum.

Therefore, it was understood that this toxin acts only at the level of borer larvae and, for this reason, it was defined as bio-insecticide.

Subsequently, several other cry proteins were characterized, all with marked bio-insecticidal properties against Lepidoptera, Diptera or beetles.

Reverse genetics and the Bt gene

Reverse genetics is the process with which we can identify the gene of interest starting from a known protein. So, at this point, it was used to identify the gene that encodes the Cry protein in B. thuringiensis.

In particular, in this technique the amino acids of a protein of interest are sequenced, then, regions containing aminoacids without many redundant codons are identified and, based on this, oligonucleotides are designed and used in the amplification by means of PCR of the gene from the chromosome. Later, this gene and the toxin it produces was called Bt.

Once it was possible to clone this gene, it was first subjected to organisms that produce large quantities of this protein, resulting in the formation of real canned bioinsecticides (Fig. 4) containing powders derived from the freeze-drying of Bt toxins.

bioinsetticida GMO
Figure 4 – Bioinsecticides [credits:Β starkbros]

Among the various advantages of the Bt protein, there is first of all the fact that if ingested by the human, the human intestine would use it like any other protein and, therefore, it would not be toxic or harmful in any way.

Furthermore, as for the environment, it does not create pollution issues as this protein has, among other things, a short half-life.

GMOs and restriction enzymes

The second step that led to the production of the first GMO is represented by restriction enzymes.

We are talking about enzymes belonging to the class of hydrolases, which catalyze the endonucleolytic cleavage of DNA at specific palindromic sites.

In this case, a restriction site is palindromic because it has sequences that repeat on the two filaments with different orientations.

The nomenclature of these enzymes depends on the bacterium of origin, for example EcoR1 for Escherichia coli (restriction 1, Fig. 5).

sito di restrizione di E. coli
Figure 5 – Restriction site of E. coli [credits:Β chimicaonline]

A restriction enzyme can have four, six, or even eight base pairs sites; the most frequent are six base pairs, such as EcoR1.

Restriction and modification mechanisms

The chromosome of the restriction enzyme producer (for example E. coli) is preserved from being cut on itself by a methylation mechanism.

In particular, a specific methylase associated with the typical restriction enzyme, in conjunction with DNA duplication, recognizes and modifies the restriction sites by methylation; thus avoiding the self-digestion of the self chromosome.

During evolution, bacteria have retained these enzymes so dangerous to deal with phage infections.

In fact, in the event that a bacterium is infected by a phage, there will be restriction sites on both the bacterial and viral chromosomes. However, while the restriction sites on the bacterial genome will be methylated and, therefore, protected by that bacterium’s restriction enzymes, those present on the viral chromosome will remain “uncovered”.

Thus, the bacterial restriction enzyme will be able to digest the viral chromosome, blocking the phage infection in the bud. Thus, this restriction and modification mechanism constitutes a primitive bacterial immune system for defense against viruses.

Hence, the presence of restriction enzymes restricts the phage population still capable of infecting a given bacterial cell.

Use of restriction systems in the industrial and medical fields

To protect as much as possible a bacterium of industrial or medical interest from phage lysis from the restriction and modification system typical of that bacterium, other restriction and modification systems coming from different bacterial strains are added.

In this way, the strain of interest will be protected not only by phages with the self sequence for their own restriction system but also by those that have sequences recognized by all the other restriction systems.

Concluding the discussion of this second step in the history of GMOs, we remind you that today it is thanks to cutting using restriction enzymes that it is possible to insert, for example, the human insulin gene in a plasmid; the latter, inserted in specific bacterial sites, will cause insulin to be produced starting, therefore, from GMO bacteria.

Original article “Il mondo degli OGM: dalla scoperta all’analisi (parte 1)” by Giovanna Spinosa

Translation by Umberto Lazzaro

Sources:

  • https://www.sciencedirect.com/science/article/pii/S0261219420302027;
  • https://dialnet.unirioja.es/servlet/articulo?codigo=7660280;
  • https://www.microbiologiaitalia.it/didattica/organismi-geneticamente-modificati-ogm/.

Image credits

  • Figura 1: https://www.informatoreagrario.it/difesa-e-fertilizzazione/difesa/mais-difesa-piralide-diabrotica-contenere-micotossine/;
  • Immagine 2: https://slideplayer.it/slide/2765215/;
  • Figura 3: https://it.wikipedia.org/wiki/Delta-endotossina#/media/File:Bt_toxin.jpg;
  • Immagine 4: https://www.starkbros.com/products/tools-and-supplies/pest-and-disease-controls/bonide-thuricide-bt;
  • Figura 5: https://www.chimica-online.it/biologia/enzimi-di-restrizione.htm.

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