Minimal cell

The surfaces in Fig. 4(d,e,f,) obtained from the functional (1) are the surfaces already discovered by Schoen [28] and named by him O, C-TO, I-WP, F-RD. O, C-TO is the only structure which cannot be minimized with respect to the cell length. For all the structures except this one we are able to find the minimal cell length i.e., by varying the cell length we are able to find the length for which the free energy per unit volume (functional (1)) has a minimum. The O, C-TO structure collapses to I-WP when the cell... 

This suggests that cyclin A2 is not essential for the early embryonic cell cycles. Also D-type cyclins seem to be dispensable for the early mouse embryo cell cycle progression since embryonic stem (ES) cells do not express them at all before differentiation (Savatier et al 1996). We do not know, however, whether the D-type cyclins are also absent in the early embryo. These observations suggest that not only could the first cell cycles of the mouse embryo have specific modifications, but also further embryonic cell cycles are specifically modified as well. Mammalian embryonic cell cycles are probably modified often during development. Such studies could allow us to determine a profile of a minimal cell cycle in mammals which must, however, be much more complex than a simple S M phase embryonic cell cycle of amphibians or insects. 

The research dealing with models for the first primitive cells has had one central topic for many years the minimal cell . According to Luisi et al. (2006a), this is defined as an artificial or semi-artificial cell which contains a minimal (but sufficient) number of components to keep the cell alive . The cell is considered to be living when three conditions are fulfilled ... 

This definition does not identify one special structure it is a descriptive term for a variety of minimal cells. Although there is no consensus on this definition, scientists do agree on the main points. Thus, a minimal genome should have 200-300 genes. The question arises as to whether it is conceivable that this number can be further reduced, or whether we need to devise other, as yet unknown, precursor systems. 

The minimal cell, as the simplest system which has all the required properties of life (metabolism, self-reproduction and the ability to evolve), is presently studied as part of a new research discipline synthetic biology. This includes subjects such as synthesis in branches of biological systems, for example, of new RNA species, new peptides and new nucleic acid analogues, as well as the synthesis of peptide nucleic acids. One example is the work of M. R. Ghadiri and G. von Kiedrowski on self-replication of oligonucleotides and oligopeptides (Luisi, 2006b). 

Poly(A) synthesis also occurred in the second system, but the product remained within the vesicles. Walde also determined the increase of the vesicle concentration, which corresponds to that expected for an autocatalytic process. In this experiment, the enzyme PNPase is first captured by the vesicle envelope, and in the second step, ADP and oleic anhydride are added the anhydride is hydrolysed to the acid. ADP passes through the vesicle double layer and is polymerized in the interior of the vesicle by PNPase to give poly(A). Hydrolysis of the anhydride causes a constant additional delivery of vesicle-forming material, so that the amount of vesicle present increases during the poly(A) synthesis. These experiments demonstrated a model for a minimal cell. Autocatalytically synthesised giant vesicles could be prepared under similar conditions and observed under a microscope (Wik et al., 1995). 

Table 9.1 Properties of an ideal minimal cell (according to Andrew Pohorille and David Deaner)...

Sensing of encapsulation-induced physiological stress will help in the devising of synthesis procedures that will minimize cell inactivation during immobilization. 

Kuruma, Y., Nishiyama, K., Shimizu, Y., Muller, M. and Ueda, T. Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins. Biotechnol Prog 21 1243-1251, 2005. 

The primary antibody in NGG-sap-PBS (e.g., 10 pg/mL mouse monoclonal antibody) is then added to the fixed cells in NGG-sap-PBS and incubated for 30 min at room temperature. Do not pipet directly onto the cells, but add antibody solutions at the edge of the dish, and add wash solutions from a wide-mouth bottle or beaker to minimize cell disattachment. The minimum volume to cover a 35-mm dish surface completely is 1 mL. 

Figure 10.5 A liposome that builds its own membrane with the help of entrapped enzymes is the prototype of the simplest autopoietic minimal cell (E, A, and S as defined in Figure 10.3).

Figure 11.1 The semi-synthetic approach to the construction of the minimal cell.

The point of this procedure is not to synthesize a fully fledged modem cell, but the simplest possible form of it. To clarify this point, the notion of the minimal cell needs to be discussed is more detail. 

One of the earliest attempts to describe the DNA/proteins minimal cell was by Morowitz (1967). Based on the enzymatic components of primary metabolism, Morowitz estimated that the size of a minimal cell should be about one-tenth smaller than mycoplasma. There were earlier significant insights to the field by Dyson (1982) as well as by Woese (1983) and Jay and Gilbert (1987). More recently, the reviews by Deamer and coworkers (Pohorille and Deamer, 2002) together with other researchers (Ono and Ikegami, 2000 Luisi, 2002a Oberholzer and Luisi, 2002) have sharpened the question and brought it into the perspective of the modern molecular tools. 

This brings us to the notion of the minimal cell, defined as that having the minimal and sufficient number of components to be called alive. What does alive mean Well, here we should go back to Chapter 2 and the various definitions of life however, a fairly general definition can be used here, which ought to keep everybody satisfied living at the cellular level means the concomitance of three properties self-maintenance (metabolism), self-reproduction, and evolvability (see Figure 11.2). 

If all these three properties are fulfilled, we have fully fledged cellular life. Of course in semi-synthetic systems the implementation can be less than perfect, and then several kinds of approximation to cellular life can be envisaged. For example, we can have protocells capable of self-maintenance but deprived of selfreproduction or vice versa. Or we can have protocells in which self-reproduction is active for only a few generations or systems that do not have the capability to evolve. Even in a given type of minimal cell - for example one with all three attributes - there might be quite different ways of implementation. 

It is clear then that the term minimal cell depicts large families of possibilities. The question is how the minimal cells can be constructed in the laboratory. From the operational point of view, the illustrations of Figures 11.1 and 11.2 already... 

Mushegian and Koonin (1996) calculated an inventory of 256 genes that represents the amount of DNA required to sustain a modern type of minimal cell under permissible conditions. This number, as indicated later by Koonin (2000), is quite similar to the values of viable minimal genome sizes inferred by site-directed gene disruptions in B. subtilis (Itaya, 1995) and transposon-mediated... 

Figure 11.4 The hypothetical pathway for the transformation of a simple RNA cell into a minimal DNA/protein cell. At the first step, the cell contains two ribozymes, Rib-1 and Rib-2 Rib-1 is a RNA replicase capable of reproducing itself and making copies of Rib-2, a ribozyme capable of synthesizing the cell membrane by converting precursor A to surfactant S. During replication, Rib-1 is capable of evolving into novel ribozymes that make the peptide bond (Rib-3) or DNA (Rib-4). In this illustration, these two mutations are assumed to take place in different compartments, which then fuse with each other to yield a protein/DNA minimal cell. Of course, a scheme can be proposed in which both Rib-3 and Rib-4 are generated in the same compartment. (Modified fromLuisi et al., 2002.)...

Table 11.4. A list of genes that define minimal cells, sorted by functional category...

All these considerations may help to decrease the number of genes down to a happy number of, say, 45-50 genes - see last column of Table 11.4 - for a hving, although certainly limping, minimal cell (Luisi et al, 2002). 

The consideration of the minimal cell permits a logical link with the notion of compartments outlined in the previous chapter. Suppose that these 45-50 macromolecules - or their precursors - developed first in solution. In order to start cellular life, compartmentation should have come later on, and one would then have to assume the simultaneous entrapment of all these different genes in the same vesicle. This can indeed be regarded as highly improbable. A more reasonable scenario is one in which the complexity of cellular life evolved from within the same compartment - a situation namely where the 45 (or 206) macromolecules were produced and evolved from a much smaller group of components from the inside of the protocell. How, of course, remains to be seen. 

The road map to the minimal cell. 1 Complex biochemical reactions in vesicles... 

Such a research project is a complex enterprise and it may be useful to divide up the road map to the minimal cell into different milestones of increasing complexity. The first one, which is already under control in several laboratories, is to carry out and optimize complex enzymatic reactions in liposomes - such as the polymerase chain reaction, the biosynthesis of RNA and DNA, the condensation of amino acids, etc. 

Perhaps one of the very first examples of enzymatic reactions carried out in liposomes with the aim of building a minimal cell is the work by Schmidli et al. (1991), as already mentioned in the previous chapter (Fig. 10.5). The general idea is illustrated in Figure 11.6, whereas the biochemical pathway is illustrated in Figure 11.7. The basic idea is to have inside the liposomes the series of reactions that, starting from a relatively simple product (G3P, glycerol-3-phosphate)... 

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