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Saturday, November 24, 2012

55. Dyson's Proteins-First Model for the Origins of Life



In 1985 Freeman John Dyson wrote a little book Origins of Life, in which he argued that metabolic reproduction and replication are logically separable propositions, and that natural selection does not require replication, at least for simple creatures. In higher-level life as seen today, reproduction of cells and replication of molecules occur together. But there is no reason to presume that this was always the case. According to Dyson, it is more likely that life originated twice, with two separate kinds of organisms, one capable of metabolism without exact replication, and the other capable of replication without metabolism. At some stage the two features came together. When replication and metabolism occurred in the same creature, natural selection as an agent for novelty became more vigorous.

Eigen and Orgel had demonstrated (cf. Part 53), by two different experiments (one involving templates and the other involving enzymes), that a solution of nucleotide monomers can, under suitable conditions in the laboratory, give rise to a nucleic-acid polymer molecule (RNA) which replicates and mutates and competes with its progeny for survival. Living cells use both templates and enzymes for making RNA. This work pointed to a possible parasitic development of RNA-based life in an environment created by a pre-existing protein-based life.


During millions of years of chemical (and later also biological) evolution, the initial primitive but living cells diversified and refined their metabolic reaction pathways. In particular, they evolved the synthesis of ATP (adenosine triphosphate) through some autocatalytic reaction mechanisms (cf. Part 46). ATP is the main energy-carrying molecule in all present-day cells. ATP-carrying primitive cells had an evolutionary advantage over other, less efficient, cells. In time, other molecules like AMP (adenosine monophosphate) emerged; or perhaps AMP came first, and then ATP.



Although ATP and AMP have similar chemical structures; they play totally different roles in present-day cells. ATP is the universal biological currency for energy. AMP, on the other hand, is one of the nucleotides in the structure of the RNA molecule.

If ATP loses two of its three phosphate groups, it becomes AMP. Dyson argued that, although the primitive cells had no genetic apparatus to begin with, they were loaded with ATP molecules which could easily convert to AMP molecules. Accidentally, in one such cell which happened to be carrying AMP and other nucleotides (the ‘chemical cousins’ of AMP), the Eigen experiment for synthesizing RNA happened spontaneously. With some help from pre-existing enzymes, an RNA molecule got produced. Once created, it went on replicating itself because of the proclivity of base A to hydrogen-bond with base U, and of G to hydrogen-bond with C.

Thus, RNA first appeared as a parasitic disease in the cell. Although most such cells died of disease, some evolved to survive the infection, à la Lynn Margulis (cf. Part 51). In such cells, the parasite gradually became a symbiont. Further evolution resulted in a situation in which the protein-based life learnt to make use of the ability for exact replication provided by the chemical structure of RNA.


Is it really true that proteins emerged before RNA? The early evidence came from laboratory experiments done during the 1950s. The well-known experiments by Miller and others (cf. Part 53) demonstrated that amino acids form easily in a reducing atmosphere from the still simpler molecules, in the presence of ultraviolet radiation.  What about nucleotides?


They are more difficult to synthesize. A nucleotide has three parts: an organic base, a sugar, and a phosphate ion. The phosphate ion occurs naturally as a constituent of rocks and sea water.  The sugar (ribose) part can be synthesized with substantial efficiency from formaldehyde. And the synthesis of an organic base was demonstrated by Oró in 1960. He prepared a concentrated solution of ammonium cyanide in water, and just let it stand. Adenine was self-created, with a 0.5% yield. Guanine also got synthesized in a similar way. But the catch here is that it is difficult to imagine how such high degrees of concentration of ammonium cyanide could occur in Nature, although some possible scenarios have been suggested.

Dyson has given an updated version of his earlier ideas, in the book Life: What a Concept!(2008). In his updated model there are six stages in the evolution of chemical complexity, leading to the emergence of life.

Stage 1. The early cells were just little bags of some kind of cell membrane (as I have described in the lipid-first model in Part 54). This is the ‘garbage bag model’ for Stage 1. And inside the bag there was a more or less random collection of organic molecules, with the characteristic that small molecules could diffuse in through the vesicle membrane, but big molecules, once synthesized, could not diffuse out. Thus the ‘garbage bag’ situation was conducive to the conversion and retention of small molecules into large molecules. The higher concentration of organic material in the bag led to a higher efficiency of the chemical processes involved.



This evolution did not involve any replication processes. ‘When a cell became so big that it got cut in half, or shaken in half, by some rainstorm or environmental disturbance, it would then produce two cells which would be its daughters, which would inherit, more or less, but only statistically, the chemical machinery inside.’

Stage 2. Parasitic RNA appeared in some of the cells in Stage 2. ATP had appeared in one of the garbage bags by a random process in Stage 1, and the cell hosting it had a metabolic advantage over other cells. Therefore many cells with large amounts of ATP got created. Then, again by chance, ATP changed to AMP in one of the cells. In due course, AMP and its chemical cousins polymerized into a primitive form of RNA. Thus there was parasitic RNA inside these cells, forming a separate form of life, which was pure replication without metabolism. To quote Dyson: ‘Then the RNA invented viruses. RNA found a way to package itself in a little piece of cell membrane, and travel around freely and independently. Stage two of life has the garbage bags still unorganized and chemically random, but with RNA zooming around in little packages we call viruses carrying genetic information from one cell to another. That is my version of the RNA world’.

Stage 3. This stage started when the protein and the RNA systems started to collaborate. This happened after the emergence of the ribosome. Although this arrangement had the rudiments of the modern cell, the genetic information was shared mostly via viruses travelling from cell to cell. This was some kind of open-source heredity. The chemical inventions made by one cell could be shared with others. Evolution went on in parallel in many different cells. The best chemical devices could be shared between different cells and combined, so the chemical evolution was very rapid, as it occurred in parallel by many pathways.

Stage 4. Speciation and sex appeared in Stage 4, and that marked the beginning of the Darwinian era, when species appeared. ‘Some cells decided it was advantageous to keep their intellectual property private, to have sex only with themselves or with the members of their own species, thereby defining species. That was then the state of life for the next two billion years, the Archeozoic and Proterozoic eras. It was a rather stagnant phase of life, continued for two billion years without evolving fast.’

Stage 5. Multicellular organisms appeared in Stage 5, which also involved death.

Stage 6. This is the stage when we humans appeared.

Saturday, November 17, 2012

54. The RNA-World Model for the Origin of Life

Modern living organisms depend on both DNA and proteins. But what came first, DNA or proteins? DNA has the codes for the synthesis of proteins, but it itself needs proteins (enzymes) for its creation. A way out of this chicken-or-egg dilemma has been found by the currently popular RNA-world model for the origin of life. According to it, since RNA (a close cousin of DNA in terms of structure etc.) has been observed to act both as an information-storage (replicating) molecule and as an enzyme, life probably started as 'nude replicating RNA molecules'.

This model of the origin of life became popular after of the discovery, made in the mid-1980s by Thomas Cech and coworkers, that certain RNA sequences called ribozymes can act as enzymes, i.e. catalyze reactions. This dual functionality of RNA might have allowed for the existence of an 'RNA species' that could replicate itself and thus seed the beginning of biomolecular evolution.



RNA is indeed known to be involved in a number of fundamental cell-biology processes. All biological cells contain ribosomes, which comprise of as much as ~60% ribosomal RNA (rRNA); the rest ~40% is protein.


Moreover, the ribosome machinery is almost identical throughout the living world; perhaps it existed almost from the beginning of life on Earth. This is an important clue because all life is believed to have had a common ancestor.



According to one version of the RNA-world hypothesis, a different type of nucleic acid, namely pre-RNA, was the first to emerge as a self-reproducing molecule, and was replaced by RNA only later. But it is also true that activated pyrimidine ribonucleotides (two of the four nucleotides constituting RNA are pyrimidines) have been synthesized in the laboratory under reasonable prebiotic conditions.

However, several scientists have expressed reservations about the RNA-world model, as discussed, for example, in a recent Wikipedia article. I shall quote the objections of Stuart Kauffman, author of the 1995 book At Home in the Universe, and the 2000 book Investigations:
  • It is difficult to get RNA strands to reproduce in a test tube. ‘No one has succeeded in achieving experimental conditions in which a single-stranded DNA or RNA could line up free nucleotides, one by one, as complements to a single strand, catalyze the ligation of the free nucleotides into a second strand, melt the two strands apart, then enter another replication cycle. It just has not worked’.
  • Even if life did tend to originate and evolve by the RNA route, naked RNA molecules must have suffered an ‘error catastrophe’ during the replication processes, thus corrupting the genetic message from generation to generation. In present-day cells, such errors (mutations) are kept to a minimum by the so-called ‘proofreading’ and ‘editing’ enzymes. (Such considerations form part of a new subject called 'systems biology'.)
  • RNA-based life, even if it did emerge, was not complex enough to sustain itself. In other words, it was too far from the edge of chaos where complexity thrives best. Why viruses do not have independent life? Why is it that the simplest free-living cells are the so-called pleuromona, and nothing less complex than them? Pleuromona are the simplest known bacteria, and they are complete with cell membrane, genes, RNA, protein-synthesizing machinery, proteins. All free-living cells have at least the minimal molecular diversity of pleuromona. Why nothing simpler exists that is alive on its own? The nude RNA or the nude ribozyme polymerase idea for the origin of life offers no decent explanation for the observed minimum necessary complexity of any life form.


I shall discuss an alternative model of the origin of life in the next post. It is based on the 'metabolism-first' idea (as opposed to the replication-first idea inherent in the RNA-world model). There are many variations of the metabolism-first idea; i.e. metabolism without genes. For example there is the lipids-first model (Segre & Lancet 2000), as sketched in Column B in the figure below. Column A depicts the RNA-first idea.


www90.homepage.villanova.edu/lowell.gustafson/scsc/lifebegin.ppt

Phospholipids are known to form lipid bilayers in water, the same structure as in cell membranes. And this is what I wrote in Part 45 about vesicles:


When a giant vesicle, which happens to have a smaller vesicle inside it, is exposed to octyl glucoside, the smaller vesicle can pass through the outer membrane into the external medium (‘birthing’). The resulting injury to the membrane of the host vesicle heals immediately. Addition of cholic acid, on the other hand, induces a feeding frenzy in which a vesicle grows rapidly as it consumes its smaller neighbours. After the food is gone, the giant vesicle then self-destructs (a case of ‘birth, growth, and death’). Such lifelike morphological changes were obtained by using commercially available chemicals; thus these processes should be assigned to organic chemistry, and not to biology or even biochemistry.

The main idea of the lipids-first model invoking vesicles is that initially information was stored in the molecular composition of lipid bodies, and the evolution of more efficient information-storage molecules like RNA and DNA occurred in a later epoch.


Saturday, November 10, 2012

53. How Life Emerged out of Nonlife



Life as we know it depends on the availability of certain long molecules (polymers): proteins, RNA, DNA, polysaccharides, lipid bilayers, etc. And the chemistry observed is all organic, i.e. carbon-based.


We have to explain three stages in the emergence of life on Earth:

Stage I. Formation of organic molecules ('monomers') from inorganic matter.

Stage II. Covalent bonding (polymerization) of the monomers into long biomolecules.

Stage III. Emergence of the complete biological cell, ~3.5 billion years ago, capable of survival and propagation.

The first major theory for explaining Stage I was put forward by Alexander Oparin in 1924. He argued that certain organic molecules that were necessary building blocks for the emergence of life required an oxygen-free or 'reducing' ambience for their self-synthesis. He imagined a 'primeval soup' of organic molecules that got created in the oxygen-free atmosphere prevailing at that time, aided by the action of sunlight. And these molecules combined in increasingly complex ways, till they formed the so-called 'coacervate' droplets. Such droplets could 'grow' by fusion with other droplets. And they could 'reproduce' through fission into smaller droplets. This primitive metabolism underwent Darwinian-like chemical evolution in which factors favouring cell integrity survived and evolved. No DNA-type replication was involved in this earliest of 'life forms'.

Similarly, J. B. S. Haldane argued that the prebiotic oceans of the Earth were very different from what they are now, and that a 'hot dilute soup' existed in them in which organic molecules could get formed.

A famous experiment for verifying the Oparin-Haldane hypothesis was carried out by Miller and Urey in 1952. They created electric sparks in a mixture of water, hydrogen, methane, and ammonia. After one week a full 10-15% of the carbon in the mixture was found to have converted to organic compounds, including amino acids (the building blocks of proteins). Recent analysis of their saved vials has revealed that as many as 23 amino acids were formed, whereas they detected only five.

Sidney Fox carried out several experiments that confirmed the formation of long organic molecules from inorganic matter. He allowed amino acids to dry out as if from a warm puddle, mimicking prebiotic conditions. The amino acids were found to form long, even cross-linked, thread-like molecules, now called 'proteinoids'. He also collected volcanic material from a cinder cone in Hawaii, and found that the temperature was over 100oC just four inches beneath the surface. He speculated that this was probably the environment in which life emerged. Biomolecules formed in such conditions could have got washed away to the seas. In an experiment, he placed lumps of lava over amino acids created from a mixture of methane, ammonia and water, sterilized the whole thing, and baked it for a few hours in a glass oven. What he got on the surface was a brown sticky substance. On drenching the lava in a sterilized water tank, a brown liquid leached out. The amino acids had formed proteinoids which, in turn, had combined to form small, cell-like spheres. These 'microspheres' are now known as 'protobionts'. These were not biological cells; they did not contain any functional nucleic acids. But they did form lumps and chains, rather like what cyanobacteria do.

Manfred Eigen and Leslie Orgel demonstrated that a solution of nucleotide monomers can, under suitable conditions in the laboratory, give rise to a nucleic-acid polymer molecule (RNA) which replicates and mutates and competes with its progeny for survival. For achieving this, Eigen used a polymerase enzyme, a protein catalyst extracted from a 'bacteriophage' (the synthesis and replication of the RNA depends on the structural guidance provided by this enzyme).

Orgel did something complementary to the experiment of Eigen. He made RNA grow out of nucleotide monomers by adding a template for the monomers to copy, but did not add a polymerase enzyme.

Thus Eigen made RNA using an enzyme but no template, and Orgel made RNA using a template but no enzyme. Modern-day living cells use both templates and enzymes for making RNA. This work pointed to a possible parasitic development of RNA-based life in an environment created by a pre-existing protein-based life.


There is a strong chance that life appeared on Earth during the so-called 'thermophilic energy regime' (I shall explain this terminology in a later post). This form of life comprised of microorganisms that thrived in hot conditions. Emergence of any form of life means a build-up of order, complexity, and information. For this to have become possible, there had to be conditions far from equilibrium, i.e. an energy gradient (cf. Part 6). One view is that, most probably, sunlight did not play a major role in this, and that the energy source was of geothermal origin. Volcanic heat sources under the sea (hydrothermal vents) provided the upper end of the energy gradient, the lower end being the cold atmosphere above the seas. Under the dominant influence of the driving force provided by this energy gradient, chemical evolution and diversification of molecular structure occurred. As proposed by Kauffman in 1993, such chemical evolution led to the emergence of autocatalytic reactions. His model obviates the need for the prior presence of information-rich DNA molecules for the synthesis of protein molecules, and also provides a non-random mechanism for the origin of life. Closed-loop autocatalytic reactions led to the production of life-like molecules of increasing complexity. Things progressed to a point where the forebears of DNA started appearing, which had the potential for replication. The biological prokaryotic cell emerged in due course.


There is also a viewpoint that, because of the presumed unfeasibility of the time scales involved for a terrestrial origin of life, life might have originated elsewhere in the cosmos, and brought to our Earth by meteors etc.

There is still a lot of debate on what really happened that led to the emergence of life on Earth. In the next two posts I shall describe two major models about the origin(s) of life.

Saturday, November 3, 2012

52. Epigenetics



When Darwin published his theory of biological evolution, the field of genetics had not yet taken shape. Naturally, Lamarck, whose work on evolution preceded that of Darwin, was also unaware of the crucial role genetic mechanisms play in the evolution of species. We now have the familiar concepts of 'genotype' and 'phenotype'. The term genotype refers to the genetic blueprint encoded in the DNA chains. Phenotype, on the other hand, signifies the characteristics manifested by an organism; it is the structure created by the organism from the instructions in its genotype.
 
In phase-space language, genotypes correspond to the search space, and phenotypes to the solution space.

Biological evolution is generally believed to be Darwinian, or rather neo-Darwinian. Lamarck’s evolution model, or Lamarckism, on the other hand, was based on two premises: the principle of use and disuse; and the principle of inheritance of acquired characteristics (without involving the genotype).

The Lamarckian viewpoint is not acceptable because it runs counter to the central dogma of molecular biology (cf. Part 43). The field of research called epigenetics has brought us close to Lamarckism in a superficial sort of way, but without violating the central dogma of molecular biology. It is now clear that changes other than those in the sequence of nucleotides in DNA, acquired during the lifetime of parents or grand-parents, can sometimes be inherited in the next few generations. Gene expression or interpretation (cf. Part 48) can be influenced by molecules hitchhiking on gametes (germ cells) and therefore on genes. This temporarily heritable hitchhiking of the genes is called epigenetic inheritance.




Genes are portions of the long sequence of nucleic acids in the DNA chain, and contain coded information for synthesizing the various proteins. And we have known since the days of Mendel that not all genes are active all the time. Some genes are dominant, while others are recessive. One of the Mendelian laws of genetics is that it is the combination of dominant (or switched on) and recessive (or switched off) genes, inherited from the parents, that dictates the characteristics (phenotype) of an offspring. We saw in Part 48 how the presence of certain chemicals can influence gene expression, and once a gene has been switched on, it acts as a switch which can alter the ‘on’ or ‘off’ states of other genes. Hormones are one example of what can influence gene activity.


Can chemicals other than hormones, for example those in the diet of an organism, also influence gene expression? The answer is 'Yes'. And not just food, but even the mental state of an creature can be responsible for the secretion of chemicals which can influence gene expression.

But from the point of view of genetics and transmittal of acquired characteristics, the influence on the transmittal mechanism of genes should be of an irreversible, perpetual nature; only then can it affect the future generations in a permanent manner; then only can we have an inheritance of acquired characteristics by the progeny. This is not found to be the case. Epigenetic transmission of acquired characteristics does not last for a significantly large number of generations.


Epigenetic effects influence the phenotype over a few generations, without changing the sequence of nucleotides along the DNA chain.

One particular heritable marking of DNA that has been investigated substantially is that of methylation, i.e. attachment of the -CH3 group to one or more nucleotides along the DNA chain.



It has been found, for example, that methylation is quite frequent in cancer cells.

Methylation affects gene expression and, as a feedforward mechanism, can have serious transgenerational effects. Epigenetic changes can be passed through the germ line for a few generations. And epigenetic changes can occur throughout the life time of an individual: Methylation/demethylation can turn some genes off/on.


Richard Dawkins' phrase 'The Selfish Gene' sums up the essence of neo-Darwinistic evolution; at least the Dawkins version of it: Natural selection acts only on genes, via their expression in an organism's body and behaviour. The genes are not naked. The phenotype is the vehicle that promotes their interest of propagating to the next generation. The organism is the survival machine for the genes. Dawkins spoke of the 'selfish gene' because the gene promotes its own survival, without necessarily promoting the survival of the organism, group or even species.

There are two aspects of a gene. The specific sequence of the four nucleic-acid bases (A, T, G, C) along the backbone of DNA; and the paraphernalia of interactions that occur on the peripheral surface of the long DNA molecule. The backbone part does not change, except through the occasional mutation, or during 'chromosomal crossover'. Epigenetics is all about what happens on the surface, with no influence on the backbone sequence of the nucleotides.

Genes propagate over many generations with a high degree of integrity because the replication process involves the strongly bonded backbone structure of DNA. By contrast, the epigenetic alterations are of a temporary (few-generations) nature; there is no evidence that the changes are permanent. In view of this, Dawkins has responded to the claims of epigenetics by replacing his phrase 'selfish gene' by 'selfish replicator':

"The 'transgenerational' effects now being described are mildly interesting, but they cast no doubt whatsoever on the theory of the selfish gene". . ."Whether [epigenetic marks] will eventually be deemed to qualify as 'selfish replicators' will depend upon whether they are genuinely high-fidelity replicators with the capacity to go on forever. This is important because otherwise there will be no interesting differences between those that are successful in natural selection and those that are not."

Dawkins points out that if the epigenetic effects fade out within the first few generations, they cannot be said to be positively selected.