Modern Techniques Make the Experimental Exploration of Evolution Possible
I. The Molecular Design of Life 7. Exploring Evolution 7.4. Evolutionary Trees Can Be Constructed on the Basis of Sequence Information Figure 7.21. The Lamprey. A jawless fish whose ancestors diverged from bony fish approximately 400 million years ago, the lamprey contains hemoglobin molecules that contain only a single type of polypeptide chain. [Brent P. Kent.] I. The Molecular Design of Life 7. Exploring Evolution 7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible Two techniques of biochemistry have made it possible to examine the course of evolution more directly and not simply by inference. The polymerase chain reaction (Section 6.1.5) allows the direct examination of ancient DNA sequences, releasing us, at least in some cases, from the constraints of being able to examine existing genomes from living organisms only. Molecular evolution may be investigated through the use of combinatorial chemistry, the process of producing large populations of molecules en masse and selecting for a biochemical property. This exciting process provides a glimpse into the types of molecules that may have existed in the RNA world. 7.5.1. Ancient DNA Can Sometimes Be Amplified and Sequenced The tremendous chemical stability of DNA (Section 2.2.7) makes the molecule well suited to its role as the storage site of genetic information. So stable is the molecule that samples of DNA have survived for many thousands of years under appropriate conditions. With the development of PCR methods, such ancient DNA can sometimes be amplified and sequenced. This approach has been applied to mitochondrial DNA from a Neanderthal fossil estimated at between 30,000 and 100,000 years of age found near Düsseldorf, Germany, in 1856. Investigators managed to identify a total of 379 bases of sequence. Comparison with a number of the corresponding sequences from Homo sapiens revealed between 22 and 36 substitutions, considerably fewer than the average of 55 differences between human beings and chimpanzees over the common bases in this region. Further analysis suggested that the common ancestor of modern human beings and Neanderthals lived approximately 600 million years ago. An evolutionary tree constructed by using these and other data revealed that the Neanderthal was not an intermediate between chimpanzees and human beings but, instead, was an evolutionary "dead end" that became extinct (Figure 7.22). Note that earlier studies describing the sequencing of much more ancient DNA such as that found in insects trapped in amber appear to have been flawed; contaminating modern DNA was responsible for the sequences determined. Successful sequencing of ancient DNA requires sufficient DNA for reliable amplification and the rigorous exclusion of all sources of contamination. 7.5.2. Molecular Evolution Can Be Examined Experimentally Evolution requires three processes: (1) the generation of a diverse population, (2) the selection of members based on some criterion of fitness, and (3) reproduction to enrich the population in more fit members (Section 2.2). Nucleic acid molecules are capable of undergoing all three processes in vitro under appropriate conditions. The results of such studies enable us to glimpse how evolutionary processes might have generated catalytic activities and specific binding abilities important biochemical functions in all living systems. A diverse population of nucleic acid molecules can be synthesized in the laboratory by the process of combinatorial chemistry, which rapidly produces large populations of a particular type of molecule such as a nucleic acid. A population of molecules of a given size can be generated randomly so that many or all possible sequences are present in the mixture. When an initial population has been generated, it is subjected to a selection process that isolates specific molecules with desired binding or reactivity properties. Finally, molecules that have survived the selection process are allowed to reproduce through the use of PCR; primers are directed toward specific sequences included at the ends of each member of the population. As an example of this approach, consider an experiment that set a goal of creating an RNA molecule capable of binding adenosine triphosphate and related nucleotides. Such ATP-bonding molecules are of interest because they might have been present in the RNA world. An initial population of RNA molecules 169 nucleotides long was created; 120 of the positions differed randomly, with equimolar mixtures of adenine, cytosine, guanine, and uracil. The initial synthetic pool that was used contained approximately 1014 RNA molecules. Note that this number is a very small fraction of the total possible pool of random 120-base sequences. From this pool, those molecules that bound to ATP, which had been immobilized on a column, were selected (Figure 7.23). The collection of molecules that were bound well by the ATP affinity column was allowed to reproduce by reverse transcription into DNA, amplification by PCR, and transcription back into RNA. This new population was subjected to additional rounds of selection for ATP-binding activity. After eight generations, members of the selected population were characterized by sequencing. Seventeen different sequences were obtained, 16 of which could form the structure shown in Figure 7.24. Each of these molecules bound ATP with high affinity, as indicated by dissociation constants less than 50 µ M. The folded structure of the ATP-binding region from one of these RNAs was determined by nuclear magnetic resonance (Section 4.5.1) methods (Figure 7.25). As expected, this 40-nucleotide molecule is composed of two Watson-Crick basepaired helical regions separated by an 11-nucleotide loop. This loop folds back on itself in an intricate way to form a deep pocket into which the adenine ring can fit. Thus, a structure was generated, or evolved, that was capable of a specific interaction. I. The Molecular Design of Life 7. Exploring Evolution 7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible Figure 7.22. Placing Neanderthal on an Evolutionary Tree. Comparison of DNA sequences revealed that Neanderthal is not on the line of direct descent leading to Homo sapiens but, instead, branched off earlier and then became extinct. I. The Molecular Design of Life 7. Exploring Evolution 7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible Figure 7.23. Evolution in the Laboratory. A collection of RNA molecules of random sequences is synthesized by combinatorial chemistry. This collection is selected for the ability to bind ATP by passing the RNA through an ATP affinity column (Section 4.1.3). The ATP-binding RNA molecules are released from the column by washing with excess ATP, and replicated. The process of selection and replication is then repeated several times. The final RNA products with significant ATP-binding ability are isolated and characterized. I. The Molecular Design of Life 7. Exploring Evolution 7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible Figure 7.24. A Conserved Secondary Structure. The secondary structure shown is common to RNA molecules selected for ATP binding.