LOC 390443 (RNase 9) on chromosome 14q11.2 is related to the RNase A superfamily and contains a unique amino-terminal preproteinlike sequence

A new member of the human RNase A superfamily is reported. Identified in the human genome assembly as LOC 390443, this locus is located 128 kb telomeric to the established RNase A gene family cluster on chromosome 14q11.2. The amino acid sequence of this locus is sufficiently similar to the eight previously identified gene family members to warrant a designation as RNase 9. RNase 9 is expressed in a wide range of human tissues. In addition, a 30-amino acid sequence lying between a 26-amino acid putative signal peptide and the last 148 amino acids that align with the other RNases A is not seen in other members of the RNase A superfamily in any species. Nucleotide and amino acid sequences of RNase 9 in 13 nonhuman primate species were determined and indicate several conserved sites but, also, an excess of nonsynonymous substitutions, about one-third of which are radical substitutions. This suggests that RNase 9, similar to several other human RNases A, has been under diversifying selection in the primates. Data from the mouse and rat genomes indicate that RNase 9 is also present in rodents, thus making it older than most of the established members of the human RNase A superfamily. Many of the human RNases A have been shown to have antimicrobial, antiviral, or antiparasitic functions involved in host-defense mechanisms. The features of RNase 9 described here suggest that it, too, may be involved in host defense and that it, along with the rest of the superfamily, may prove to have played an important role in anthropoid evolution.     

Duplication and divergence of 2 distinct pancreatic ribonuclease genes in leaf-eating African and Asian colobine monkeys

Unique among primates, the colobine monkeys have adapted to a predominantly leaf-eating diet by evolving a foregut that utilizes bacterial fermentation to breakdown and absorb nutrients from such a food source. It has been hypothesized that pancreatic ribonuclease (pRNase) has been recruited to perform a role as a digestive enzyme in foregut fermenters, such as artiodactyl ruminants and the colobines. We present molecular analyses of 23 pRNase gene sequences generated from 8 primate taxa, including 2 African and 2 Asian colobine species. The pRNase gene is single copy in all noncolobine primate species assayed but has duplicated more than once in both the African and Asian colobine monkeys. Phylogenetic reconstructions show that the pRNase-coding and noncoding regions are under different evolutionary constraints, with high levels of concerted evolution among gene duplicates occurring predominantly in the noncoding regions. Our data suggest that 2 functionally distinct pRNases have been selected for in the colobine monkeys, with one group adapting to the role of a digestive enzyme by evolving at an increased rate with loss of positive charge, namely arginine residues. Conclusions relating our data to general hypotheses of evolution following gene duplication are discussed.     

Investigating ancient duplication events in the Arabidopsis genome

The complete genomic analysis of Arabidopsis thaliana has shown that a major fraction of the genome consists of paralogous genes that probably originated through one or more ancient large-scale gene or genome duplication events. However, the number and timing of these duplications still remains unclear, and several different hypotheses have been put forward recently. Here, we reanalyzed duplicated blocks found in the Arabidopsis genome described previously and determined their date of divergence based on silent substitution estimations between the paralogous genes and, where possible, by phylogenetic reconstruction. We show that methods based on averaging protein distances of heterogeneous classes of duplicated genes lead to unreliable conclusions and that a large fraction of blocks duplicated much more recently than assumed previously. We found clear evidence for one large-scale gene or even complete genome duplication event somewhere between 70 to 90 million years ago. Traces pointing to a much older (probably more than 200 million years) large-scale gene duplication event could be detected. However, for now it is impossible to conclude whether these old duplicates are the result of one or more large-scale gene duplication events. abbreviations dA, fraction of amino acid substitutions; Kn, number of nonsynonymous substitutions per nonsynonymous site; Ks, number of synonymous substitutions per synonymous site; MYA, million years ago     

Positive selection and propeptide repeats promote rapid interspecific divergence of a gastropod sperm protein

Male-specific proteins have increasingly been reported as targets of positive selection and are of special interest because of the role they may play in the evolution of reproductive isolation. We report the rapid interspecific divergence of cDNA encoding a major acrosomal protein of unknown function (TMAP) of sperm from five species of teguline gastropods. A mitochondrial DNA clock (calibrated by congeneric species divided by the Isthmus of Panama) estimates that these five species diverged 2-10 MYA. Inferred amino acid sequences reveal a propeptide that has diverged rapidly between species. The mature protein has diverged faster still due to high nonsynonymous substitution rates (> 25 nonsynonymous substitutions per site per 10(9) years). cDNA encoding the mature protein (89-100 residues) shows evidence of positive selection (Dn/Ds > 1) for 4 of 10 pairwise species comparisons. cDNA and predicted secondary-structure comparisons suggest that TMAP is neither orthologous nor paralogous to abalone lysin, and thus marks a second, phylogenetically independent, protein subject to strong positive selection in free-spawning marine gastropods. In addition, an internal repeat in one species (Tegula aureotincta) produces a duplicated cleavage site which results in two alternatively processed mature proteins differing by nine amino acid residues. Such alternative processing may provide a mechanism for introducing novel amino acid sequence variation at the amino-termini of proteins. Highly divergent TMAP N-termini from two other tegulines (Tegula regina and Norrisia norrisii) may have originated by such a mechanism.     

Timing the origin of New World monkeys

The origin of New World monkeys (Infraorder Platyrrhini) has been an extensively debated issue. In this study, we analyzed mitochondrial genomes from Cebus (Platyrrhini), Homo, Hylobates, Pan, Pongo (Hominoids), Macaca, Papio (Cercopithecoids), and Tarsius (outgroup) to investigate this matter. Two distinct methodologies were employed on mitochondrial genes to estimate divergence times: the traditional likelihood ratio test performed in ML analyses of individual and concatenated gene sequences and the recent multigene Bayesian approach. Using the Cercopithecoid-Hominoid split as calibration point (25 MYA), our results show consistently that Platyrrhines split from Catarrhines at around 35 MYA. Although the main focus of the study is New World monkey origins, we have also estimated other primate divergence times: Homo-Pan at 5-7 MYA; Pongo-(Homo/Pan) at 13-16 MYA; Hylobates-(Pongo/Homo/Pan) at 15-19 MYA; and Macaca-Papio at 10-12 MYA. Our estimate for the origin of New World monkeys is in agreement with the hypothesis of a transatlantic journey from Africa to South America, as suggested by the fossil record.     

A comparative study on the catalytic properties of guanyl-specific ribonucleases.

The kinetic parameters of reactions catalyzed by four guanyl-specific RNases T1, Pb1, Th1 and Sa were studied comparatively using three types of substrates; guanosine-2',3'-cyclophosphates, GpN dinucleoside phosphates and synthetic polyribonucleotides. The kinetic parameters were shown to be similar in spite of considerable differences in primary structures of these RNases, including amino acid residues of the active sites. Therefore, primary structures of guanyl RNases allow for a considerable number of substitutions (both in the 'recognising' and catalytical parts of the active site) without changes in the catalytical parameters.     

Parallel evolution of site‐specific changes in divergent caribou lineages

The parallel evolution of phenotypes or traits within or between species provides important insight into the basic mechanisms of evolution. Genetic and genomic advances have allowed investigations into the genetic underpinnings of parallel evolution and the independent evolution of similar traits in sympatric species. Parallel evolution may best be exemplified among species where multiple genetic lineages, descended from a common ancestor, colonized analogous environmental niches, and converged on a genotypic or phenotypic trait. Modern North American caribou (Rangifer tarandus) originated from three ancestral sources separated during the Last Glacial Maximum (LGM): the Beringian–Eurasian lineage (BEL), the North American lineage (NAL), and the High Arctic lineage (HAL). Historical introgression between the NAL and the BEL has been found throughout Ontario and eastern Manitoba. In this study, we first characterized the functional differentiation in the cytochrome‐b (cytB) gene by identifying nonsynonymous changes. Second, the caribou lineages were used as a direct means to assess site‐specific parallel changes among lineages. There was greater functional diversity within the NAL despite the BEL having greater neutral diversity. The patterns of amino acid substitutions occurring within different lineages supported the parallel evolution of cytB amino acid substitutions suggesting different selective pressures among lineages. This study highlights the independent evolution of identical amino acid substitutions within a wide‐ranging mammal species that have diversified from different ancestral haplogroups and where ecological niches can invoke parallel evolution.     

Stomach lysozyme gene of the langur monkey: Tests for convergence and positive selection

Summary Genomic blotting and enzymatic amplification show that the genome of the langur monkey (like that of other primates) contains only a single gene for lysozymec, in contrast to another group of foregut fermenters, the ruminants, which have a multigene family encoding this protein. Therefore, the langur stomach lysozyme gene has probably evolved recently (i.e., within the period of monkey evolution) from a conventional primate lysozyme. The sequences of cDNAs for the stomach lysozyme of langur and the conventional lysozymes of three other Old World monkeys were determined. Identification of the promoter for the stomach gene and comparison to the human gene, which is expressed conventionally in macrophages, show that both lysozyme genes use the same promoter. This suggests that the difference in expression patterns is due to change(s) in enhancer or silencer regulatory elements. With the cDNA sequences the hypothesis that the langur stomach lysozyme has converged in amino acid sequence upon the stomach lysozymes of ruminants is tested. Consistent with the convergence hypothesis, only those sites that specify amino acids in the mature lysozyme are shared uniquely with ruminant lysozyme genes. None of the silent sites at third positions of codons or in noncoding regions support a link between the langur and ruminants. Statistical analysis based on silent sites rules out the possibility of horizontal transfer of a stomach lysozyme gene between the langur and ruminant lineages and supports the close relationship of the langur lysozyme gene to that of other monkeys.     

Within-host evolution of CD8+-TL epitopes encoded by overlapping and non-overlapping reading frames of simian immunodeficiency virus

In order to understand the impact of overlapping reading frames on natural selection by host CD8+ T lymphocytes (CD8(+)-TL), we analyzed the pattern of nucleotide substitution in simian immunodeficiency virus (SIV) genomes sampled from populations at time of death in 35 rhesus monkeys. Both the mean number of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and the mean number of synonymous nucleotide substitutions per synonymous site (d(S)) were elevated in overlap regions in comparison to non-overlap regions. Mean d(N) exceeded mean d(S) in CD8(+)-TL epitopes restricted by the host's class I major histocompatibility complex molecules. This pattern, which is indicative of positive Darwinian selection favoring amino acid changes in these epitopes, was seen in both overlap and non-overlap regions; but mean d(N) was particularly elevated in restricted CD8(+)-TL epitopes encoded in overlap regions. Amino acid changes from the inoculum were defined as parallel if the same amino acid change occurred at the same site independently in two or more monkeys, and a surprisingly high proportion (71.9%) of observed amino acid changes throughout the SIV genome occurred in parallel in different monkeys. The proportion of parallel changes in restricted epitopes encoded by overlapping reading frames was still higher (80%), supporting the hypothesis that the interaction of positive selection and overlapping reading frames enhances the probability of convergent or parallel amino acid change.     

Simultaneous positive and purifying selection on overlapping reading frames of the tat and vpr genes of simian immunodeficiency virus

Tat-specific cytotoxic T cells have previously been shown to exert positive Darwinian selection favoring amino acid replacements of an epitope of simian immunodeficiency virus (SIV). The region of the tat gene encoding this epitope falls within a region of overlap between the tat and vpr reading frames, and nonsynonymous nucleotide substitutions in the tat reading frame were found to occur disproportionately in such a way as to cause synonymous changes in the vpr reading frame. Comparison of published complete SIV genomes showed Tat to be the least conserved at the amino acid level of nine proteins encoded by the virus, while Vpr was one of the most conserved. Numerous parallel amino acid changes occurred within the Tat epitope independently in different monkeys, and purifying selection on the vpr reading frame, by limiting acceptable nonsynonymous substitutions in the tat reading frame, evidently has enhanced the probability of parallel evolution.     

Functional decline of sweet taste sensitivity of colobine monkeys

For many primates, sweet taste is palatable and is an indicator that the food contains carbohydrates, such as sugars and starches, as energy sources. However, we have found that Asian colobine monkeys (lutungs and langurs) have low sensitivity to various natural sugars. Sweet tastes are recognized when compounds bind to the sweet taste receptor TAS1R2/TAS1R3 in the oral cavity; accordingly, we conducted a functional assay using a heterologous expression system to evaluate the responses of Javan lutung (Trachypithecus auratus) TAS1R2/TAS1R3 to various natural sugars. We found that Javan lutung TAS1R2/TAS1R3 did not respond to natural sugars such as sucrose and maltose. We also conducted a behavioral experiment using the silvery lutung (Trachypithecus cristatus) and Hanuman langur (Semnopithecus entellus) by measuring the consumption of sugar-flavored jellies. Consistent with the functional assay results for TAS1R2/TAS1R3, these Asian colobine monkeys showed no preference for sucrose or maltose jellies. These results demonstrate that sweet taste sensitivity to natural sugars is low in Asian colobine monkeys, and this may be related to the specific feeding habits of colobine monkeys.     

Primary structural features of rosaceous S-RNases associated with gametophytic self-incompatibility

We isolated cDNA clones encoding five S-RNases (S_1-,_S3- , S_5-, S_6-, S_7-RNases) from pistils of Pyrus pyrifolia (Japanese pear), a member of the Rosaceae. Their amino acid sequences were aligned with those of other rosaceous S-RNases sequenced so far. A total of 76 conserved amino acid residues were stretched throughout the sequence, but were absent from the 51–66 region which was designated the hypervariable (HV) region. The phylogenetic tree of rosaceous S-RNases showed that S-RNase polymorphism predated the divergence of Pyrus and Malus. Pairwise comparison of these S-RNases detected two highly homologous pairs, P. pyrifolia S_1- and S_4-RNases (90.0%) and P. pyrifolia S_3- and S_5-RNases (95.5%). The positions of amino acid substitutions between S_1- and S_4-RNases were spread over the entire region, but in the pair of S_3- and S_5-RNases, amino acid substitutions were found in the 21–90 region including the HV region. The substitutions in this restricted region appear to be sufficient to discriminate between S₃ and S₅ pollen and to trigger the self-incompatible reaction.     

Mitochondrial data support an odd-nosed colobine clade

To obtain a more complete understanding of the evolutionary history of the leaf-eating monkeys we have examined the mitochondrial genome sequence of two African and six Asian colobines. Although taxonomists have proposed grouping the "odd-nosed" colobines (proboscis monkey, douc langur, and the snub-nosed monkey) together, phylogenetic support for such a clade has not been tested using molecular data. Phylogenetic analyses using parsimony, maximum likelihood, and Bayesian methods support a monophyletic clade of odd-nosed colobines consisting of Nasalis, Pygathrix, and Rhinopithecus, with tentative support for Nasalis occupying a basal position within this clade. The African and Asian colobine lineages are inferred to have diverged by 10.8 million years ago (mya or Ma). Within the Asian colobines the odd-nosed clade began to diversify by 6.7 Ma. These results augment our understanding of colobine evolution, particularly the nature and timing of the colobine expansion into Asia. This phylogenetic information will aid those developing conservation strategies for these highly endangered, diverse, and unique primates.     

Pattern of Nucleotide Substitutions in Growth Hormone-Prolactin Gene Family: A Paradigm for Evolution by Gene Duplication

The growth hormone-prolactin gene family in mammals is an interesting example of evolution by gene duplication. Divergence among members of duplicated gene families and among species was examined by using reported gene sequences of growth hormone, prolactin and their receptors. Sequence divergence among species was found to show a general tendency in which a generation-time effect is pronounced for synonymous substitutions but not so for nonsynonymous substitutions. Divergence among duplicated genes is characterized by the relatively high rate of nonsynonymous substitutions, i.e., the rate is close to that of synonymous ones. In view of the stage- and tissue-specific expression of duplicated genes, some of the amino acid substitutions among duplicated genes is likely to be caused by positive Darwinian selection.     

Characterization of Ribonucleases from Culture Medium of Lentinus edodes

Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate. The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom. RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2. In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified. Since RNase Le2, which has very similar N-terminal sequence to RNase Le 37 and RNase Le 45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism.     

Simian immunodeficiency virus mutants resistant to serum neutralization arise during persistent infection of rhesus monkeys.

We previously described the pattern of sequence variation in gp120 following persistent infection of rhesus monkeys with the pathogenic simian immunodeficiency virus SIVmac239 molecular clone (D.P.W. Burns and R.C. Desrosiers, J. Virol. 65:1843, 1991). Sequence changes were confined largely to five variable regions (V1 to V5), four of which correspond to human immunodeficiency virus type 1 (HIV-1) gp120 variable regions. Remarkably, 182 of 186 nucleotide substitutions that were documented in these variable regions resulted in amino acid changes. This is an extremely nonrandom pattern, which suggests selective pressure driving amino acid changes in discrete variable domains. In the present study, we investigated whether neutralizing-antibody responses are one selective force responsible at least in part for the observed pattern of sequence variation. Variant env sequences called 1-12 and 8-22 obtained 69 and 93 weeks after infection of a rhesus monkey with cloned SIVmac239 were recombined into the parental SIVmac239 genome, and variant viruses were generated by transfection of cultured cells with cloned DNA. The 1-12 and 8-22 recombinants differ from the parental SIVmac239 at 18 amino acid positions in gp120 and at 5 and 10 amino acid positions, respectively, in gp41. Sequential sera from the monkey infected with cloned SIVmac239 from which the 1-12 and 8-22 variants were isolated showed much higher neutralizing antibody titers to cloned SIVmac239 than to the cloned 1-12 and 8-22 variants. For example, at 55 weeks postinfection the neutralizing antibody titer against SIVmac239 was 640 while those to the variant viruses were 40 and less than 20. Two other rhesus monkeys infected with cloned SIVmac239 showed a similar pattern. Rhesus monkeys were also experimentally infected with the cloned variants so that the type-specific nature of the neutralizing antibody responses could be verified. Indeed, each of these monkeys showed neutralizing-antibody responses of much higher titer to the homologous variant used for infection. These experiments unambiguously demonstrate that SIV mutants resistant to serum neutralization arise during the course of persistent infection of rhesus monkeys.     

Pleistocene diversification of living squirrel monkeys (Saimiri spp.) inferred from complete mitochondrial genome sequences

In order to enhance our understanding of the evolutionary history of squirrel monkeys (Saimiri spp.), we newly sequenced and analyzed data from seven complete mitochondrial genomes representing six squirrel monkey taxa. While previous studies have lent insights into the taxonomy and phylogeny of the genus, phylogenetic relationships and divergence date estimates among major squirrel monkey clades remain unclear. Using maximum likelihood and Bayesian procedures, we inferred a highly resolved phylogenetic tree with strong support for a sister relationship between Saimiri boliviensis and all other Saimiri, for monophyly of Saimiri oerstedii and Saimiri sciureus sciureus, and for Saimiri sciureus macrodon as the sister lineage to the S. oerstedii/S. s. sciureus clade. We inferred that crown lineages for extant squirrel monkeys diverged around 1.5 million years ago (MYA) in the Pleistocene Epoch, with other major clades diverging between 0.9 and 1.1 MYA. Our results suggest a relatively recent timeline of squirrel monkey evolution and challenge previous conceptions about the diversification of the genus and its expansion into Central America.     

Pancreatic-Type Ribonuclease 1 Gene Duplications in Rat Species

Mammalian pancreatic-type ribonucleases (RNases) 1 represent single-copy genes in the genome of most investigated mammalian species, including Mus musculus and other murid rodents. However, in six species belonging to the genus Rattus and closely related taxa, several paralogous gene products were identified by Southern blotting and PCR amplifications of genomic sequences. Phylogenies of nucleotide and derived amino acid sequences were reconstructed by several procedures, with three Mus species as outgroup. Duplications of the RNase 1 occurred after the divergence of Niviventer cremoriventer and Leopoldamys edwardsi from the other investigated species. Four groups of paralogous genes could be identified from specific amino acid sequence features in each of them. Low ratios of nonsynonymous-to-synonymous substitutions and the paucity of pseudogene features suggest functional gene products. One of the RNase 1 genes of R. norvegicus is expressed in the pancreas. RNases 1 were isolated from pancreatic tissues of R. rattus and R. exulans and submitted to N-terminal amino acid sequence analysis. In R. rattus, the orthologue of the expressed gene of R. norvegicus was identified, but in R. exulans, two paralogous gene products were found. The gene encoding for one of these had not yet been found by PCR amplification of genomic DNA. A well-defined group of orthologous sequences found in five investigated species codes for very basic RNases. Northern blot analysis showed expression of messenger RNA for this RNase in the spleen of R. norvegicus, but the protein product could not be identified. Evolutionary rates of RNase 1, expressed as nucleotide substitutions per site per 10(3) million years (Myr), vary between 5 and 9 in the lines leading to Mus, Niviventer, and Lepoldamys (on the basis of an ancestral date of mouse/rat divergence of 12.2 Myr) and between 20 and 50 in the lines to the other sequences after divergence from Niviventer and Leopoldamys (5.5 Myr).     

Uniformity of colour vision in Old World monkeys

It is often assumed that all Old World monkeys share the same trichromatic colour vision, but the evidence in support of this conclusion is sparse as only a small fraction of all Old World monkey species have been tested. To address this issue, spectral sensitivity functions were measured in animals from eight species of Old World monkey (five cercopithecine species and three colobine species) using a non-invasive electrophysiological technique. Each of the 25 animals examined had spectrally well-separated middle- and long-wavelength cone pigments. Cone pigments maximally sensitive to short wavelengths were also detected, implying the presence of trichromatic colour vision. Direct comparisons of the spectral sensitivity functions of Old World monkeys suggest there are no significant variations in the spectral positions of the cone pigments underlying the trichromatic colour vision of Old World monkeys.     

Functional promiscuity of squirrel monkey growth hormone receptor toward both primate and nonprimate growth hormones

Primate growth hormone (GH) has evolved rapidly, having undergone approximately 30% amino acid substitutions from the inferred ancestral eutherian sequence. Nevertheless, human growth hormone (hGH) is physiologically effective when administered to nonprimate mammals. In contrast, its functional counterpart, the human growth hormone receptor (hGHR), has evolved species specificity so that it responds only to Old World primate GHs. It has been proposed that this species specificity of the hGHR is largely caused by the Leu --> Arg change at position 43 after a prior His --> Asp change at position 171 of the GH. Sequence analyses supported this hypothesis and revealed that the transitional phase in the GH:GHR coevolution still persists in New World monkeys. For example, although the GH of the squirrel monkey has the His --> Asp substitution at position 171, residue 43 of its GHR is a Leu, the nonprimate residue. If the squirrel monkey truly represents an intermediate stage of GH:GHR coevolution, its GHR should respond to both hGH and nonprimate GH. Also, if the emergence of species specificity was a result of the selection for a more efficient GH:GHR interaction, then changing residue 43 of the squirrel monkey growth hormone receptor (smGHR) to Arg should increase its binding affinity toward higher primate GH. To test these hypotheses, we performed protein-binding assays between the smGHR and both human and rat GHs, using the surface plasmon resonance methodology. Furthermore, the effects of reciprocal mutations at position 43 of human and squirrel monkey GHRs are measured for their binding affinities toward human and squirrel monkey GHs. The results from the binding kinetic assays clearly demonstrate that the smGHR is in the intermediate state of the evolution of species specificity. Interestingly, the altered residue Arg at position 43 of the smGHR does not lead to an increased binding affinity. The implications of these results on the evolution of the GH:GHR interaction and on functional evolution are discussed.