This text represents only a theoretical supposition and has three parts. In the first part I explain a hypothetical implication of introns in cell differentiation and the consecutive genetic mechanism of this participation.

In the second part I have tried to show the dynamic processes of cell differentiation and the relation between cell determination and cell differentiation.

In the third part I explain these theories with a concrete example of differentiating model: the very early commitment of the embryonic cells to trophoblast or inner cell mass

 

Part I. INTRONS IN CELL DIFFERENTIATION

            The histonic proteins are those that fold the DNA, awarding a stable structure but not an accessible one to translation of information in RNA. Preparation for translating of DNA chain is made by modifying the conformation of this, as a result of the interaction between nonhistonical proteins and the introns sequences of the genes that are going to be activated.

A certain type of cell (for example muscular cell) contains a certain set of nonhistonic proteins- type 1. This type wills interact with a sequence of DNA – x sequence, (Fig. 1) which is repeating and is found at the level of introns in a lot of active genes of the muscular cell. (For ex. genes which codify miozyn, actine, mioglobin etc.).

Into another type of cell (for example liver cell) there is another kind of nonhistonical proteins - type 2 (Fig. 1). This one will interact with y sequence, which is found at the level of introns in a lot of active genes of the liver cell. This is a very simple model because in every cell exists a large number of nonhistonical proteins which can activate a very large number of genes.

            The problem is how can be realized the different expression of genes, referring to the fact that structure of DNA chain is the same in all cells.  This problem is especially important in the initial phases of ontogenesis, when certain extrinsically factors (which influences differentiation of diverse cellular populations), do not exist. So, I have tried to explain the process of cellular differentiation using some hypothetical intrinsically genetic mechanisms.

  Fig. 1

Cell synthesis, in G1 phase, two different types of nonhistonical proteins: types 1 and types 2 (Fig.1). These are in an inactive state and are spread aleatory in the entire cell. Still in this phase cell synthesis other two types of proteins, called interface proteins: type A and type B, and two enzymatic systems. Each one of these interface proteins is make up of two components: an identical fragment for both, which is complementary with a part of DNA, called X site, and another fragment which is complementary with the corresponding fragment of the other interface protein. So, these two types of protein molecules will be coupled two by two by helping of the complementary regions (Fig. 1). When starts the synthesis of DNA (S phase), each chain will have attached, at X-site level, a certain type of interface protein (the connection will be made aleatory). Although in both cells DNA chain has the same structure, because of those two different types of proteins, this will achieve a different conformation (that’s the reason I have called these two protein types ‘interface proteins’).

Let’s suppose in one daughter cell (cell A), at X-sites DNA’s level, it is attached interface protein A. This one, by helping of complementary fragment (which have several enzymatic sites), will activate one of two enzymatic systems (which are found aleatory, in both daughter cells, just like nonhistonic proteins A and B). The result is the activation of nonhistonic protein types 1 (although, in both daughter cells, it can be found both nonhistonic protein type 1 and 2). The nonhistonic protein type 1 will attaches to the repeating x sequence, which is part of introns of a certain set of genes. These genes will be activated (Fig. 1).

The other daughter cell (cell B), will have attached on X site of DNA, interface protein type B. This, by helping of the other enzymatic system will activate nonhistonic protein type 2. This will be attached into a specific mode to another repeatable y sequence (Fig. 1), which is localized at the level of introns of another set of genes that are going to be activated. So, one could explain, how two different cellular lines can be spontaneously formed from a Susa cell, by genetic mechanisms and without implying another extrinsically factors.

 

Part II. THE RELATION BETWEEN DETERMINATION AND DIFFERENTIATION

 

             To explain certain phenomenon which appears during the first stages of ontogenesis, we must introduce the notion called cellular determination. The state of determinate cell is an intermediate one, which follows to the state of a pluripotent cell and which will precedes the state of differentiated cell.

In the first stage appear two types of determined cells: type A and type B, which are not different from a morphological point of view but only because of the potential of activating different sets of genes (Fig. 2).

For instance, cell A according to the previous theory, contains nonhistonical active protein type I. This will activate the gene a, which synthesize receptors of membrane (R) and soluble receptors in the extra cellular space and the gene b, which synthesize another type of inactive nonhistonical protein – type II (Fig.4).

  Fig. 4

In the following stage the type A cells, spread in the cellular population (resulting from repeated divisions of the pluripotent cells), will migrate one to another due to receptors interactions (Fig. 2). As the type A cells interaction, the activation of receptors of membrane determines the activation, through specific mediators, of nonhistonical protein initially synthesized – type II, (Fig.4). This will activate the genes c and d, which will synthesize the specific proteins as well that gene e, which is responsible by its own synthesis. In this moment takes place the cellular differentiation. Now the cells of type A are different from the cells of type B (by morphological point of view) being grouped and forming a distinctive embryonic structure.

Thus, a determinate cell has two variants (Fig.2):

                        1. As a result of interaction (through a direct contact or at a distance by helping of soluble receptors) with the other cells of the same type (cell A with cell A and cell B with cell B) is initiated the process of cellular differentiation resulting in forming a certain type of embryonic structure (or later a certain type of tissue);

                        2. If it is not realized this contact then the process of differentiation does not takes place and the determined cell may come back at the state of pluripotency, after some divisions, because the nonhistonical protein (type I) synthesized in the moment of cellular determination (Figure 4), does not activate the gene which is reponsable by its own synthesis. At the moment of cellular differentiation (Figure 4) nonhistonic protein type II does intensify the own synthesis, so the phenomenon of cellular differentiation becomes irreversible.

             In this way, an important law of biology is reflected at the molecular level, namely the ontogeny is a short and quick revision of the ontogeny. The process of cellular differentiation is a sequential one and it progress step by step. Every realized stage is the beginning for a new stage, and the activation of the divers’ sets of genes, which have appeared during the evolution in a chronological order, is based on this process.

 

       How can maintain a cellular line a state of differentiation?

In normal condition, a cellular line will maintain a differentiation state during the cellular multiplication if the nonhistonic protein activate, about the genes which are responsible by the morfofunctional specific features of the respective cellular line, also the gene which are responsible by its own synthesis (otherwise, that nonhistonic proteins will arrive at a minimum concentration after several divisions). Thus it is created a phenomenon of a positive feed-back (Figure 3).

 

 

Part III. The early mammalian embryo

 

 

 

I send you a concrete example of differentiating model: the very early commitment of the embryonic cells to trophoblast or inner cell mass.

The early development of mammalian embryo has, from this point of view, three stages.

            1. Stage one: up to the eight cell stage, each blastomere is still totipotent and can go to form at least both foetus and trophoblast;

2. Stage two (a temporary stage) from 8 to 16 cell stage, when distinction between inside and outside cells first becomes manifest suggesting that it is this event which begins to limit totipotency. But in this stage at least some blastomeres remains totipotent, and if remove from the embryo or displace within, it may alter their ultimate fate.

            3. Stage three: above 32 cells stage of development: the trophoblast and inner cell mass are differentiated and determined; The distinction between trophoblast and inner cell mass are represented by biochemical parameters and surface cell receptors (when isolated and grown in culture the outer cells are found to form closed multicellular vesicles; cells from the inside of the blastocyst never show this property)

The fecundation moment start synthesis of two types of nonhistonic proteins (in inactive state) and of interface proteins (which are connected two by two)-see Fig. 1. The differentiation begins when the interface protein connect with a hypothetic X site.       Because there is only one site, the probability of connecting depends on the concentration level of interface protein. In the very early stage the embryonic volume did not grow too much, so the probability of connecting depends almost entirely on the number of mythosis. So, after 3-4 mythosis (8-16 cell stage) the concentration of interface protein is high enough to make the connecting between interface protein and X site (Fig. 1). Each of two daughter cells will have different nonhistonic protein (as an active protein-see Figure 1) and will active different genes. These will synthesize different receptors for each of two types of cells and different proteins for chemotaxis and cellular movement (Fig. 2). The inner cells receptors are synthesized earlier, so that these cells are located inside the blastocyst. This stage is the stage two: these two types of cells are not actually determined because the nonhistonic proteins are only temporary activated; these are unstable proteins and these are quickly inactivated. So, in this stage, it is very important for every cell to move to and interacts with the same type of cell to form a compact population made up by the same type of cells (see Fig. 2 and Fig. 3). This is a crucial moment: the interaction between the same type of receptors will start the synthesis of nonhistonic proteins (as an active protein), like a positive feed-back regulation (see Fig. 3). In this way the state of differentation are maintain and the cells are determined (stage three: above 32-64 cell stage).

Each of these proteins will activate the genes which synthesizing the nonhistonic proteins of the next differentation step. And so on to the higly differentiated cells, in which specific nonhistonic proteins will activate (connecting with specific introns sequences-see Fig. 1) the specific genes for a certain tissue. The interface protein is the same, but will be synthesized by allelic genes. The only differences is how fast will be synthesized this protein. This will decide, as we could see, when the differentiating phenomenon will start.

An important mechanism will be the inductor factors (proteins and mRNA, as external factors) that appear in the next step of development. This is very important but it is not the only one mechanism, which can induce the differentation. The intrinsic mechanism must be present even in the latter stages of differentiation (when the very specialized tissues will appear) The ability of the original embryonic cell of teratoma and of stem cell line of teratocarcinomas to differentiate into a variety of identifiable highly differentiated cell type in the very limited environment of the tumora site sustain this assertion.

 

FEW ARGUMENTS TO PROVE THE VALIDITY OF THIS THEORY

1. First argument: introns are found only in pluricellular, differentiate, organisms. Beside, there is a tight connection between differentiation degree of one organism and proportion of introns in the genome of the same organism.

2. At the bacterium level expression of genes is controlled by connecting   certain proteins with certain repetitive sequences of the bacterial genome placed near the gene that is going to be activated.

Thus the role of these primitive repetitive sequences will be taking over, during the evolution, at the level of differentiated organisms, by the intronics sequences.

The existence of the repetitive sequences helps to the interaction with goveming proteins, these having the role of epitop, the interaction between these being similar to the interaction between an antigen and an anticorp.

3. Another argument could be the main laws of the biology: ontogenesis represents a short and hasty summing up of the philogenesis.

4. The existence of theratoms may be another argument that props up the theory I have already displayed. They are tumors which proceed from the multiplication of one germinate cells. Thus appears areas of smooth muscular texture, bone, cartilage, hair etc.

If expression of each gene is controlled by one gene, then, a large of simultaneous mutations would be necessary, for the appearance of this type of teratoma. But this fact is unlikely. In accordance with this theory it is necessary to appear few mutation at the level of a reduce number of genes, which synthesize nonhistonical proteins, and could express concomitantly a large number of genes specific for a certain texture. In accordance with this model, the existence of theratomas becomes most likely.

 

 

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