# Introduction

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Sequence Reconstruction Example

# History

A computer scientist at the University of Southern California named Leonard Adleman first came up with the idea of using DNA for computing purposes after reading "Molecular Biology of the Gene," a book written by James Watson (co-discoverer of the structure of DNA).

In 1994 Adleman published an article in the journal Science (L. M. Adleman, Science 266, 1021–102; 1994) that proposed how DNA could be used to solve a well-known mathematical problem, called the directed Hamilton Path problem, also known as the "traveling salesman" problem. The goal of the problem is to find the shortest path between a number of cities, going through each city only once.

## Pipeline for Generating Ancestral Genes

1. Sequences from extant species of the desired common ancestral gene and outgroup genes are aligned.
2. The ancestral gene is inferred based on evolutionary models (typically maximum parsimony or maximum likelihood).
3. Ancestral genes are synthesized via overlapping oligonucleotides and PCR assembly.
4. Ancestral genes are cloned and tested for function.

### Methods of Inferring Ancient Sequences

• Consensus Sequence - the most frequently occurring residue of a extant organisms is assumed to be the ancestral state.
• Maximum Parsimony - minimization of the total number of changes required to account for the terminal sequences.
• Maximum Likelihood - ancestral states are evaluated at each internal node in the tree based on the likelihood of each mutation. This process uses a statistical framework of molecular evolution which takes into account differences in certain types of mutations. The generated ancestral sequence gives the probability that each residue is correctly predicted.

### Gene Synthesis

Modern advances in oligonucleotide synthesis allow for the cheap construction of synthetic ancestral genes. Using overlapping PCR assembly most synthetic genes can be constructed for several hundred dollars.

Expression of archaic proteins in extant organisms such as E. coli may require codon optimization to allow for proper expression[1]. Synthesized genes are typically cloned into a vector and used to transform a host organism.

### Testing Ancestral Variants

After synthesis is complete, ancestral genes can be tested for their function. For example, the ancestral genes can be cloned into bacterial expression vectors and transformed into E.coli. The proteins can be purified and tested for their activity, whether that be fluorescence spectra, binding affinity, thermoactivity, etc.

## Examples of Ancestral Sequence Reconstructions

As the technology for ancestral sequence reconstruction has advanced the technique has popularized. It offers a unique way to peer into the past and revisit the biomolecules of our forefathers. The sensitivity of genetic information to degrade limits our understanding to extant organisms or circumstantially preserved ones, such as specimens preserved by ice. The ability to resurrect sequences from the dead and test them relieves us from these temporal limitations.

### Evolution of Coral Pigments

Phylogenetic tree of coral pigments drawn on petri dish with bacteria expressing extant and ancestral fluorescent proteins.

One example of ancestral sequence reconstruction was done by the Matz group (currently residing at the University of Texas at Austin). Fluorescent proteins from related coral species had wavelengths corresponding to Cyan, Green, and Red[2]. The details of the evolution of fluorescent color in the GFP superfamily was not fully understand. That is, what fluorescent spectra did the common ancestors of the modern corals have?

Different models for reconstruction based on amino acids, codons and nucleotides resulted in reconstructed proteins differing in 4-8 amino acids out of 217. Gene synthesis utilized codons designed "to be degenerate in order to incorporate alternative predictions." [2] The reconstructed sequences included red, pre-red, Red/Green and ALL. The ancestral sequences revealed an interesting evolutionary history. The ancestor to all the coral fluorescent proteins had a single green peak at 505nm. Gradually, a shift towards greater expression of lower wavelengths developed, culminating in the pre-red protein. One branch of pre-red went on to lose the ancestral green peak and develop a strong peak around 580nm. Modern red features significantly less emission at outside of the primary peak, thereby increasing its specificity compared to recent ancestors such as pre-red and Red/Green. According to Ugalde et al., incremental gains in protein function "[have] been anticipated since Darwin, but has only recently been demonstrated in computer simulation experiments"

### Inferring the Paleoenvironment of ancient Earth

Ancestral sequence reconstruction has been used to infer the environmental conditions on the early Earth[3]. The study sought to explore the evolutionary history of an elongation factor thermo unstable (EFtu). This elongation factor functions optimally at the temperature which the organism lives, for example thermophilic organisms have an EFtu optimized for high temperatures. By resurrecting the EFtu protein in the common ancestors of bacteria, the temperature profile might elucidate what the environmental conditions were. Interestingly the EFtu common ancestor to all mesophilic bacterium (~1BYA) has an optimal temperature of a thermophile. This suggests that the hypothesis of a hot early Earth is true.

### Precambrian β lactamases

Comparison of the denaturation temperatures of several reconstructed β lactamases compared to extant versions. PNCA is the last common ancestor (CA) of gram-positive and gram-negative bacteria. GNCA is the CA of the gram-negative bacteria. GPBCA is the CA of Gammaproteobacteria. ENCA is the CA of Enterobacteria

The structure of the last common ancestor to extant β lactamase has been reconstructed and tested using Ancestral Sequence Reconstruction[4]. Sequences for β lactamase from 75 bacterial strains, whose last CA lived 2-3 Gya, were compared. To exclude the effects of recent antibiotic driven evolution, only non-clinical varieties were chosen. Using Bayesian statistics, Risso et al. calculated the most probabilistic ancestral amino acid for each point in the sequence.

Resistance to various β lactam based antibiotics was conferred to modern microbes engineered to produce the Precambrian enzyme. The extremely high promiscuity of the Precambrian enzyme compared to extant sequences indicates that over the past few billion years, β lactamases have evolved greater substrate specificity. The Precambrian enzyme featured a denaturation temperature of ~90°C, which is 35°C above that of the highest extant sequence.

### Precambrian Thioredoxin

Perez-Jiminez et. al resurrected various Precambrian Thioredoxins. These belonged to the last common ancestors of the last bacterial common ancestor (LBCA), the last archaeal common ancestor (LACA) and the archaeal-eukaryotic common ancestor (AECA)"[1]. All of these sequences are believed to have been present over 3.5 Gya. The amino-acid sequence in each case was determined by maximum likelihood inference from 200 extant sequences. The genes to code for these proteins were synthesized and cloned into E. coli for expression and purification.

All three reconstructed proteins showed a Tm around 113°C, with a ΔTm between the highest extant Thioredoxin and the ancestors of around 25°C. The three paleo Thioredoxins also showed substantially greater activity at pH5 than representative extant enzymes from the each domain. The lower substrate specificity exhibited by the reconstructed enzymes indicates the abundance of sulfur rich compounds in the early oceans of Earth, and also hints at the generalist nature of archaic enzymes.

## Further Applications

Ancestral sequence reconstruction holds various benefits. The most direct benefit is a better understanding of archaic organisms and the world they inhabited. Numerous ancient protein reconstructions have yielded enzymes that are much more thermally stable than extant derivatives, even those found in thermophiles, leading to stronger evidence for extremely warm global conditions prior to 1 Gya[5]. Archaic proteins also allow for greater study of evolvability, as many reconstructed proteins feature high promiscuity[4]. The patterns in the evolution of high promiscuity Precambrian β lactamase, for instance, could help further the fight against the development of antibiotic resistance. High promiscuity is also key to directed protein evolution. In addition, the reconstruction of archaic enzymes serves as a sort of paleo-bioprospecting[1], and is currently the best approach to expand thermal stability and pH tolerance in enzymes[6]. The enzymes discovered through this form of prospecting and those developed through directed evolution of promiscuous enzymes could serve functional roles in human engineered systems.