Austin L. Hughes

The Evolution of Functionally Novel Proteins after Gene Duplication

A widely cited model of the evolution of functionally novel proteins (here called the model of mutation during non-functionality (MDN model)) holds that, after gene duplication, one gene copy is redundant and free to accumulate substitutions at random. By chance, some of these substitutions may suit the protein encoded by such a non-functional gene to a new function, which it can subsequently assume. Several lines of evidence contradict this hypothesis: (i) comparison of expressed duplicate genes from the tetraploid frog Xenopus laevis suggests that such genes are subject to purifying selection and are thus not free to accumulate substitutions at random; (ii) in a number of multi-gene families, there is now evidence that functionally distinct proteins have arisen not as a result of chance fixation of neutral variants but rather as a result of positive Darwinian selection; and (iii) the phenomenon of gene sharing, in which a single gene encodes a protein having two distinct functions, shows that gene duplication is not a necessary prerequisite to the evolution of a new protein function. A model for the evolution of new protein is proposed under which a period of gene sharing ordinarily precedes the evolution of functionally distinct proteins. Gene duplication then allows each daughter gene to specialize for one of the functions of the ancestral gene. However, if the ancestral gene is not bifunctional, either of the following two outcomes is expected to follow gene duplication: (i) one copy will be silenced by a mutation preventing expression; or (ii) if both copies continue to be expressed, both will be subject to purifying selection, as a high proportion of non-synonymous mutations will have a completely or partly dominant deleterious effect.

Phylogenies of Developmentally Important Proteins Do Not Support the Hypothesis of Two Rounds of Genome Duplication Early in Vertebrate History

Abstract. It has been proposed that two rounds of duplication of the entire genome (polyploidization) occurred early in vertebrate history (the 2R hypothesis); and the observation that certain gene families important in regulating development have four members in vertebrates, as opposed to one in Drosophila, has been adduced as evidence in support of this hypothesis. However, such a pattern of relationship can be taken as support of the 2R hypothesis only if (1) the four vertebrate genes can be shown to have diverged after the origin of vertebrates, and (2) the phylogeny of the four vertebrate genes (A–D) exhibits a topology of the form (AB) (CD), rather than (A) (BCD). In order to test the 2R hypothesis, I constructed phylogenies for nine protein families important in development. Only one showed a topology of the form (AB) (CD), and that received weak statistical support. In contrast, four phylogenies showed topologies of the form (A) (BCD) with statistically significant support. Furthermore, in two cases there was significant support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes: in one case there was significant support for duplication of the vertebrate genes at least prior to the divergence of vertebrates and urochordates, and in one case there was weak support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes. Taken together with other recently published phylogenies of developmentally important genes, these results provide strong evidence against the 2R hypothesis.

Evolution of the mammalian MHC: natural selection, recombination, and convergent evolution

Summary: The genes that encode molecules involved in antigen presentation within the class I and class II regions of the mammalian major histocompatibility complex (MHC) include several that are highly polymorphic. There is evidence that this polymorphism is maintained by positive selection, most likely overdominant selection, relating to their role in presenting foreign peptides to T cells. This selection can maintain allelic lineages for much longer periods of time than neutral polymorphisms are expected to last, but sharing of polymorphic amino acid motifs among species of different mammalian orders is due to independent (or convergent) evolution rather than common ancestry. It has been suggested that interallelic recombination (gene conversion) plays a role in enhancing polymorphism, but there is evidence of striking differences among loci with respect to the rate at which such recombination has contributed to current polymorphism. Recent attempts to interpret linkage relationships in the MHC region as evidence of ancient genomic duplications are not supported by phylogenetic analysis. Rather, natural selection may have played a role in the linkage of other genes to those of the MHC.

Adaptive diversification within a large family of recently duplicated, placentally expressed genes

The pregnancy-associated glycoproteins (PAG) are putative peptide-binding proteins and products of a large family of genes whose expression is localized to the placental surface epithelium of artiodactyl species. We have tested the hypothesis that natural selection has favored diversification of these genes by examining patterns of nucleotide substitution in a sample of 28 closely related bovine, caprine, and ovine family members that are expressed only in trophoblast binucleate cells. Three observations were made. First, in codons encoding highly variable domains of the proteins, there was a greater accumulation of both synonymous and nonsynonymous mutations than in the more conserved regions of the genes. Second, in the variable regions, the mean number of nonsynonymous nucleotide substitutions per site was significantly greater than the mean number of synonymous substitutions per site. Third, nonsynonymous changes affecting amino acid charge occurred more frequently than expected under random substitution. This unusual pattern of nucleotide substitution implies that natural selection has acted to diversify these PAG molecules at the amino acid level, which in turn suggests that these molecules have undergone functional diversification. We estimate that the binucleate cell-expressed PAG originated 52 +/- 6 million years ago, soon after the divergence of the ruminant lineage. Thus, rapid functional diversification of PAG expressed in trophoblast binucleate cells seems to have been associated with the origin of this unique placental adaptation.

Natural selection on the peptide-binding regions of major histocompatibility complex molecules

The major histocompatibility complex (MHC) of vertebrates is a multigene family whose products encode cell surface glycoproteins that function to present peptides to T cells (Klein 1986). There are two major subfamilies, class I and class II, the members of which differ both structurally and functionally. Class I molecules have a nearly universal pattern of expression and present peptides to cytotoxic T cells, while class II molecules are expressed on antigen-presenting cells of the immune system. In mammals, the class I and class II loci are linked together in a single gene complex, which also includes some genes that have neither an evolutionary nor a functional relationship to the MHC genes, and others which, although not evolutionarily related to the MHC genes, encode molecules that play roles in peptide presentation. Both class I and class II molecules are heterodimers with four extracellular domains, but they achieve analogous structures in different ways. The class [ heterodimer includes an ct chain consisting of three extracellular domains (cq, a~, and ct3), a transmembrane portion, and a cytoplasmic domain. The o~3 domain associates noncovalently with ~2-microglobulin ([32m). ~32m is encoded outside the MHC gene complex but shows evidence of an evolutionary relationship with the t~3 domain, which like it is a C ltype domain belonging to the immunoglobulin (Ig) superfamily (Williams and Barclay 1988). The class II heterodimer consists of an o~ chain and a ~3 chain, each of which includes two extracellular domains (~1 and c~2 and 131 and ~3% respectively), a transmembrane region, and a cytoplasmic tail; both chains are encoded by genes within the MHC gene complex. Like the membrane-proximal domains of the

Widespread purifying selection at polymorphic sites in human protein-coding loci

Estimation of gene diversity (heterozygosity) at 1,442 single-nucleotide polymorphism (SNP) loci in an ethnically diverse sample of humans revealed consistently reduced gene diversities at SNP loci causing amino acid changes, particularly those causing amino acid changes predicted to be disruptive to protein structure. The reduction of gene diversity at these SNP loci, in comparison to SNPs in the same genes not affecting protein structure, is evidence that negative natural selection (purifying selection) has reduced the population frequencies of deleterious SNP alleles. This, in turn, suggests that slightly deleterious mutations are widespread in the human population and that estimation of gene diversity even in a sample of modest size can help guide the search for disease-associated genes.

A Dominant Role for CD8+-T-Lymphocyte Selection in Simian Immunodeficiency Virus Sequence Variation

ABSTRACT CD8+ T lymphocytes (CD8-TL) select viral escape variants in both human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. The frequency of CD8-TL viral escape as well as the contribution of escape to overall virus diversification has not been assessed. We quantified CD8-TL selection in SIV infections by sequencing viral genomes from 35 SIVmac239-infected animals at the time of euthanasia. Here we show that positive selection for sequences encoding 46 known CD8-TL epitopes is comparable to the positive selection observed for the variable loops of env. We also found that >60% of viral variation outside of the viral envelope occurs within recognized CD8-TL epitopes. Therefore, we conclude that CD8-TL selection is the dominant cause of SIV diversification outside of the envelope.

Extraepitopic Compensatory Substitutions Partially Restore Fitness to Simian Immunodeficiency Virus Variants That Escape from an Immunodominant Cytotoxic-T-Lymphocyte Response

ABSTRACT Selection for escape mutant immunodeficiency viruses by cytotoxic T lymphocytes (CTL) has been well characterized and may be associated with disease progression. CTL epitopes accrue escape mutations at different rates in vivo. Interestingly, certain high-frequency CTL do not select for escape until the chronic phase of infection. Here we show that mutations conferring escape from immunodominant CTL directed against an epitope in the viral Gag protein are strongly associated with extraepitopic mutations in gag in vivo. The extraepitopic mutations partially restore in vitro replicative fitness of viruses bearing the escape mutations. Constraints on epitope sequences may therefore play a role in determining the rate of escape from CTL responses in vivo.

The MHC E locus in macaques is polymorphic and is conserved between macaques and humans

Although the functions of the molecules encoded by the classical MHC class I loci are well defined, no function has been ascribed to the molecules encoded by the non-classical MHC class I loci. To investigate the evolution and conservation of the non-classical loci, we cloned and sequenced HLA-E homologues in macaques. We isolated four E locus alleles from five rhesus monkeys and two E locus alleles from one cynomolgus monkey, which indicated that the E locus in macaques is polymorphic. We also compared the rate of nucleotide substitution in the second intron of the macaque and human E locus alleles with that of exons two and three. The rate of nucleotide substitution was significantly higher in the introns, which suggested that the E locus has evolved under selective pressure. Additionally, comparison of the rates of synonymous and non-synonymous substitutions in the peptide binding region versus the remainder of the molecule suggested that the codons encoding the amino acids in the peptide binding region had been conserved in macaques and humans over the 36 million years since macaques and humans last shared a common ancestor.

Very large long-term effective population size in the virulent human malaria parasite Plasmodium falciparum.

It has been proposed that the virulent human malaria parasite Plasmodium falciparum underwent a recent severe population bottleneck. In order to test this hypothesis, we estimated the effective population size of this species from the patterns of nucleotide substitution at 23 nuclear protein–coding loci, using a variety of methods based on coalescent theory. Both simple methods and phylogenetically based maximum–likelihood methods yielded the conclusion that the effective population size of this species has been of the order of at least 105 for the past 300 000—400 000 years.