Primordial Sequences Research Articles

Ancestral Sequence Reconstruction as a Tool for the Elucidation of a Stepwise Evolutionary Adaptation.

Ancestral sequence reconstruction (ASR) is a powerful tool to infer primordial sequences from contemporary, i.e., extant ones. An essential element of ASR is the computation of a phylogenetic tree whose leaves are the chosen extant sequences. Most often, the reconstructed sequence related to the root of this tree is of greatest interest: It represents the common ancestor (CA) of the sequences under study. If this sequence encodes a protein, one can "resurrect" the CA by means of gene synthesis technology and study biochemical properties of this extinct predecessor with the help of wet-lab experiments.However, ASR deduces also sequences for all internal nodes of the tree, and the well-considered analysis of these "intermediates" can help to elucidate evolutionary processes. Moreover, one can identify key mutations that alter proteins or protein complexes and are responsible for the differing properties of extant proteins. As an illustrative example, we describe the protocol for the rapid identification of hotspots determining the binding of the two subunits within the heteromeric complex imidazole glycerol phosphate synthase.

Divergent evolution of a bifunctional de novo protein.

Primordial proteins, the evolutionary ancestors of modern sequences, are presumed to have been minimally active and nonspecific. Following eons of selective pressure, these early progenitors evolved into highly active and specific proteins. While evolutionary trajectories from poorly active and multifunctional generalists toward highly active specialists likely occurred many times in evolutionary history, such pathways are difficult to reconstruct in natural systems, where primordial sequences are lost to time. To test the hypothesis that selection for enhanced activity leads to a loss of promiscuity, we evolved a de novo designed bifunctional protein. The parental protein, denoted Syn-IF, was chosen from a library of binary patterned 4-helix bundles. Syn-IF was shown previously to rescue two different auxotrophic strains of E. coli: ΔilvA and Δfes. These two strains contain deletions for proteins with very different biochemical functions; IlvA is involved in isoleucine biosynthesis, while Fes is involved in iron assimilation. In two separate experiments, Syn-IF, was evolved for faster rescue of either ΔilvA or Δfes. Following multiple rounds of mutagenesis, two new proteins were selected, each capable of rescuing the selected function significantly faster than the parental protein. In each case, the evolved protein also lost the ability to rescue the unselected function. In both evolutionary trajectories, the original bifunctional generalist was evolved into a monofunctional specialist with enhanced activity.

Isotypic variation of novel immunoglobulin-like transcript/killer cell inhibitory receptor loci in the leukocyte receptor complex.

The leukocyte receptor complex (LRC) on human chromosome 19q13.4 encompasses at least four families of related genes: immunoglobulin-like transcripts (ILT), killer cell inhibitory receptors (KIR), the leukocyte-associated inhibitory receptors (LAIR) and the Fcalpha receptor (Fc(alpha)R). We determined the genomic organization of a region of DNA spanning the junction of the ILT and KIR gene complexes. Extensive sequence data were collected for ILT3, two novel genes, ILT9 and ILT10, and one novel KIR locus (KIRCI). These loci, along with other reported sequences from the region, encoded a leader sequence split into two exons, upstream of two to four immunoglobulin (Ig) domains, each on a separate exon. Downstream of the Ig domains, however, the organization differs markedly between inhibitory and activating ILT. These data are consistent with a highly conserved gene arrangement for all superfamily members suggesting duplication of primordial sequences. ILT3 and KIRCI were in the same head-to-tail orientation as has been described for other KIR loci which may facilitate addition or loss of genes between different haplotypes.

Atavistic mutations reflect the long life span of dispensable genes.

Most of the major innovations in evolution occurred at the very beginning of life on this earth some 3.5 billion years ago before the division of eukaryotes from prokaryotes. This initial innovativeness was due, in no small part, to the peculiar construction of primordial coding sequences that were repeats of base oligomers, the number of bases in oligomeric units not being a multiple of three. Such coding sequences are conferred with a measure of immortality. Because of this initial immortality and of long life span of genes after becoming dispensable, the ancient gene may remain silenced in particular phylogenetic trees for a very long time, only to be resurrected later. Hemoglobin genes expressed in exceptional bacteria, plants, worms, insects, as well as in all vertebrates are a good example of this. Atavistic mutations are more dramatic visible examples of such resurrection of long dormant genes. A few interesting examples are given.

Origin of eukaryotic introns: a hypothesis, based on codon distribution statistics in genes, and its implications.

A hypothesis for the origin of introns in eukaryotic genes is developed. By computer simulation it was found that the reading-frame lengths in a random nucleotide sequence are distributed in a negative exponential manner and that there exists an upper limit of about 200 codons in the length of the reading frames (RFs). These characteristics suggest that, if primordial DNA contained a random nucleotide sequence, the most primitive cells would have been under selective pressure to eliminate interfering stop codons in order to increase the length of RFs. Further, they indicate that the only possible way that a coding sequence that is considerably longer than 600 nucleotides could be derived from the short coding sequences occurring in a random sequence would be to splice the short coding sequences and to eliminate the stretches of sequences containing clusters of inframe stop codons. Thus, introns are suggested to be those stretches of sequences containing interfering stop codons that were originally earmarked in the first primitive cells to be eliminated in order to enable the coding for long polypeptides. Because the statistical characteristics of codon distributions in today's eukaryotic DNA sequences resemble closely those of a random sequence and because the upper limit in the length of RFs (200 codons) in a random sequence corresponds precisely to the observed maximum length of exons in today's eukaryotic genes (600 nucleotides), it is suggested that introns originated in the most primitive unicellular eukaryotes when they evolved from primordial sequences. The data from the prokaryotic gene sequences indicate that prokaryotic genes may have been derived originally from primitive unicellular eukaryotic genes by losing introns from them.