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Upon vascular injury, tissue damage, ischemia, or microbial infection, intracellular material such as nucleic acids and histones is liberated and comes into contact with the vessel wall and circulating blood cells. Such “Danger-associated molecular patterns” (DAMPs) may thus have an enduring influence on the inflammatory defense process that involves leukocyte recruitment and wound healing reactions. While different species of extracellular RNA (exRNA), including microRNAs and long non-coding RNAs, have been implicated to influence inflammatory processes at different levels, recent in vitro and in vivo work has demonstrated a major impact of ribosomal exRNA as a prominent DAMP on various steps of leukocyte recruitment within the innate immune response. This includes the induction of vascular hyper-permeability and vasogenic edema by exRNA via the activation of the “vascular endothelial growth factor” (VEGF) receptor-2 system, as well as the recruitment of leukocytes to the inflamed endothelium, the M1-type polarization of inflammatory macrophages, or the role of exRNA as a pro-thrombotic cofactor to promote thrombosis. Beyond sterile inflammation, exRNA also augments the docking of bacteria to host cells and the subsequent microbial invasion. Moreover, upon vessel occlusion and ischemia, the shear stress-induced release of exRNA initiates arteriogenesis (i.e., formation of natural vessel bypasses) in a multistep process that resembles leukocyte recruitment. Although exRNA can be counteracted for by natural circulating RNase1, under the conditions mentioned, only the administration of exogenous, thermostable, non-toxic RNase1 provides an effective and safe therapeutic regimen for treating the damaging activities of exRNA. It remains to be investigated whether exRNA may also influence viral infections (including COVID-19), e.g., by supporting the interaction of host cells with viral particles and their subsequent invasion. In fact, as a consequence of the viral infection cycle, massive amounts of exRNA are liberated, which can provoke further tissue damage and enhance virus dissemination. Whether the application of RNase1 in this scenario may help to limit the extent of viral infections like COVID-19 and impact on leukocyte recruitment and emigration steps in immune defense in order to limit the extent of associated cardiovascular diseases remains to be studied.

Endothelzellen fungieren als schützende Barriere zwischen Blut und Gewebe und sind maßgeblich an der Regulation und Aufrechterhaltung der vaskulären Homöostase beteiligt. Demzufolge kann eine Fehlfunktion des Endothels auf Grund von Entzündungen, Infektionen oder Verletzungen zu einer Vielzahl von Krankheitszuständen, wie Thrombosen oder Atherosklerose, führen. In diesem Kontext wurde Ribonuklease 1 (RNase1), eine zirkulierende, extrazelluläre Endonuklease, als gefäß- und gewebeschützendes Enzym und wichtiger Regulator der vaskulären Homöostase identifiziert. Bei akuten Entzündungsprozessen fungiert RNase1 als natürlicher Gegenspieler der extrazellulären RNA (eRNA), einem „damage-associated molecular pattern“. Hierbei degradiert RNase1 akkumulierende eRNA, um die Zellen vor einer übermäßigen Entzündungsreaktion zu schützen. Bei lang anhaltenden Entzündungen kommt es jedoch zu einer Störung des RNase1-eRNA Gleichgewichtes. Dabei induziert die Akkumulation von eRNA die Freisetzung großer Mengen proinflammatorischer Zytokine durch zirkulierende Entzündungszellen, wie zum Beispiel Tumornekrosefaktor alpha (TNF-α) oder Interleukin 1 beta (IL-1β). Diese Zytokine können das Endothel entzündungsbedingt beeinflussen und führen unter Anderem zur Repression der RNase1-Expression, welche möglicherweise durch die Aktivierung von Histon-Deacetylasen (HDACs) vermittelt wird. Die hier vorliegende Studie untersuchte, ob die entzündungsvermittelte Deacetylasefunktion der HDACs durch Veränderung von Chromatinmodifikationen mit der RNase1-Repression in humanen Endothelzellen einhergeht. Dabei führte die Stimulation humaner Nabelschnurvenenendothelzellen mit TNF-α oder IL-1β zu einer signifikanten Reduktion der RNase1-Expression. Durch Identifizierung der RNASE1Promotorregion und Analysen des Chromatinstatus wurde weiterhin eine Verbindung zwischen der RNase1-Repression und der Deacetylierung von Histon 3 Lysin 27 und Histon 4 aufgezeigt. Zusätzlich konnte durch Inhibierung der Klasse I-HDACs 1-3, mittels des spezifischen Inhibitors MS275, die RNase1 mRNA sowie die damit verbundene Promotoracetylierung selbst nach Zytokinstimulation der Endothelzellen wiederhergestellt werden. Zur Identifizierung der funktionellen HDAC wurden Chromatin-Immunopräzipitations-Kinetiken durchgeführt, welche eine signifikante Akkumulation von HDAC2 am RNASE1-Promotor nach TNF-α-Stimulation aufzeigten. Diese Ergebnisse wurden durch siRNA-vermittelte Hemmung der HDAC2Expression und dessen redundanten Enzyms HDAC1 bestätigt, da auch diese Behandlung die RNase1 mRNA-Expression nach proinflammatorischer Stimulation wiederherstellen konnte. Diese Ergebnisse identifizieren HDAC2 als einen Hauptfaktor in der RNase1-Regulation in humanen Endothelzellen. Dabei wird HDAC2 nach proinflammatorischer Stimulation zum RNASE1-Promotor rekrutiert, um die Histonacetylierung zu verhindern und so die Genexpression zu reprimieren. Demzufolge eröffnet der hier aufgezeigte protektive Effekt des spezifischen HDAC-Inhibitors MS275 neuartige Therapieansätze zur Förderung der Gefäßintegrität durch Verhinderung der RNase1-Repression in entzündeten Endothelzellen., Endothelial cells (ECs) function as protective barrier to separate the blood from the surrounding tissue by conducting crucial roles in regulation and maintenance of vascular homeostasis, such as control of vessel permeability or coagulation. Therefore, dysfunction of the EC barrier due to inflammation, infection or injury can cause a variety of vascular pathologies, such as thrombosis or atherosclerosis. In this context, the circulating extracellular endonuclease Ribonuclease 1 (RNase1) was identified as a vessel- and tissue-protective enzyme and a potent regulator of vascular homeostasis. Upon acute inflammation, RNase1 functions as a natural counterpart to extracellular RNA (eRNA), a damage-associated molecular pattern, via degradation to protect the EC cell layer from excessive inflammation. However, long-term inflammation disrupts the RNase1-eRNA system. Thereby, eRNA accumulates in the extracellular space to induce massive proinflammatory cytokine release from circulating inflammatory cells, such as tumor necrosis factor alpha (TNF-α) or interleukin 1 beta (IL-1β). These cytokines negatively affect the EC layer by downregulation of RNase1 presumably through activation of histone deacetylases (HDACs). In this regard, this study investigated whether inflammation-mediated deacetylase function of HDACs suppresses RNase1 expression in human ECs through modulation of chromatin modifications. Proinflammatory stimulation with TNF-α or IL-1β of human umbilical vein endothelial cells significantly reduced RNase1 expression. Thus, identification of the RNASE1 promoter region and analysis of its chromatin state revealed the association of RNASE1 repression with deacetylation of histone 3 at lysine 27 and histone 4. The important role of HDACs in this process was further confirmed by administration of the specific class I HDAC1-3 inhibitor MS275 that successfully restored RNASE1 promoter acetylation and mRNA abundance upon TNF-α or IL-1β treatment. These results indicate an essential impact of HDAC1-3 in RNase1 regulation. Additionally, identification of specific HDACs involved in RNase1 regulation was obtained by chromatin immunoprecipitation kinetics confirming significant accumulation of HDAC2 at the RNASE1 promoter upon TNF-α stimulation. These findings were further validated by siRNA double knockdown of HDAC2 and its redundant enzyme HDAC1, which also recovered RNase1 mRNA abundance upon proinflammatory stimulation. In conclusion, our data identified HDAC2 as a crucial factor in RNase1 regulation in human ECs. HDAC2 is recruited to the RNASE1 promoter site to attenuate histone acetylation and suppress subsequent gene repression. This effect can be blocked by the specific HDAC inhibitor MS275 implicating the potential of HDAC inhibitors as novel therapeutic strategy to promote vascular integrity by preventing RNase1 downregulation in EC inflammation.

In foregut-fermenting mammals (e.g., colobine monkeys, artiodactyl ruminants) the enzymes pancreatic ribonuclease (RNASE1) and lysozyme C (LYZ), originally involved in immune defense, have evolved new digestive functions. Howler monkeys are folivorous non-colobine primates that lack the multi-chambered stomachs of colobines and instead digest leaves using fermentation in the caeco-colic region. We present data on the RNASE1 and LYZ genes of four species of howler monkey (Alouatta spp.). We find that howler monkey LYZ is conserved and does not share the substitutions found in colobine and cow sequences, whereas RNASE1 was duplicated in the common ancestor of A. palliata, A. seniculus, A. sara, and A. pigra. While the parent gene (RNASE1) is conserved, the daughter gene (RNASE1B) has multiple amino acid substitutions that are parallel to those found in RNASE1B genes of colobines. The duplicated RNase in Alouatta has biochemical changes similar to those in colobines, suggesting a novel, possibly digestive function. These findings suggest that pancreatic ribonuclease has, in parallel, evolved a new role for digesting the products of microbial fermentation in both foregut- and hindgut-fermenting folivorous primates. This may be a vital digestive enzyme adaptation allowing howler monkeys to survive on leaves during periods of low fruit availability.

Although cetaceans (whales, porpoises, and dolphins) have multi-chambered stomachs, feeding habits of modern cetaceans have dramatically changed from herbivorous to carnivorous. However, the genetic basis underlying this dietary switch remains unexplored. Here, we present the first systematic investigation of 10 digestive enzymes genes (i.e., CYP7A1, CTRC, LIPC, LIPF, PNLIP, PGC, PRSS1, SI, SLC5A1, and TMPRSS15) of representative cetaceans, and the evolutionary trajectory of RNASE1 in cetartiodactylans. Positive selections were detected with proteinases (i.e., CTRC, PRSS1, and TMPRSS15) and lipases (i.e., CYP7A1, LIPF, and PNLIP) suggesting that cetaceans have evolved an enhanced digestion capacity for proteins and lipids, the major nutritional components of their prey (fishes and invertebrates). In addition, it was found that RNASE1 gene duplicated after the cetartiodactylan speciation and two independent gene duplication events took place in Camelidae and Ruminantia. Positive selection was detected with RNASE1 of Camelidae and Bovidae, suggesting enhanced digestive efficiency in the ruminants. Remarkably, even though the ancestors of cetaceans were terrestrial artiodactyls that are herbivorous, modern cetaceans lost the pancreatic RNASE1 copy with digestive function, which is in accordance with the dietary change from herbivorous to carnivorous. In sum, this is the first study that provides new insights into the evolutionary mechanism of dietary switch in cetaceans.

The ribonuclease (RNase) A superfamily is a vertebrate-specific gene family. Because of a massive expansion that occurred during the early mammalian evolution, extant mammals in general have much more RNase genes than nonmammalian vertebrates. Mammalian RNases have been associated with diverse physiological functions including digestion, cytotoxicity, angiogenesis, male reproduction, and host defense. However, it is still uncertain when their expansion occurred and how a wide array of functions arose during their evolution. To answer these questions, we generate a compendium of all RNase genes identified in 20 complete mammalian genomes including the platypus, Ornithorhynchus anatinus. Using this, we delineate 13 ancient RNase gene lineages that arose before the divergence between the monotreme and the other mammals (∼220 Ma). These 13 ancient gene lineages are differentially retained in the 20 mammals, and the rate of protein sequence evolution is highly variable among them, which suggest that they have undergone extensive functional diversification. In addition, we identify 22 episodes of recent expansion of RNase genes, many of which have signatures of adaptive functional differentiation. Exemplifying this, bursts of gene duplication occurred for the RNase1, RNase4, and RNase5 genes of the little brown bat (Myotis lucifugus), which might have contributed to the species' effective defense against heavier pathogen loads caused by its communal roosting behavior. Our study illustrates how host-defense systems can generate new functions efficiently by employing a multigene family, which is crucial for a host organism to adapt to its ever-changing pathogen environment.

Pancreatic ribonuclease gene (RNASE1) was previously shown to have undergone duplication and adaptive evolution related to digestive efficiency in several mammalian groups that have evolved foregut fermentation, including ruminants and some primates. RNASE1 gene duplications thought to be linked to diet have also been recorded in some carnivores. Of all mammals, bats have evolved the most diverse dietary specializations, mainly including frugivory and insectivory. Here we cloned, sequenced and analyzed RNASE1 gene sequences from a range of bat species to determine whether their dietary adaptation is mirrored by molecular adaptation. We found that seven insect-eating members of the families Vespertilionidae and Molossidae possessed two or more duplicates, and we also detected three pseudogenes. Reconstructed RNASE1 gene trees based on both Bayesian and maximum likelihood methods supported independent duplication events in these two families. Selection tests revealed that RNASE1 gene duplicates have undergone episodes of positive selection indicative of functional modification, and lineage-specific tests revealed strong adaptive evolution in the Tadarida β clade. However, unlike the RNASE1 duplicates that function in digestion in some mammals, the bat RNASE1 sequences were found to be characterized by relatively high isoelectric points, a feature previously suggested to promote defense against viruses via the breakdown of double-stranded RNA. Taken together, our findings point to an adaptive diversification of RNASE1 in these two bat families, although we find no clear evidence that this was driven by diet. Future experimental assays are needed to resolve the functions of these enzymes in bats.

Ribonuclease 1 (RNase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. However, the expression patterns and potential biological function of RNase1 in herbivorous fish is not known. Here, we identified RNase1 from five fish species and illuminated the functional diversification and expression of RNase1 in herbivorous Megalobrama amblycephala. The five identified fish RNase1 genes all have the signature motifs of the RNase A superfamily. No expression of Ma-RNase1 was detected in early developmental stages but a weak expression was detected at 120 and 144 hours post-fertilization (hpf). Ma-RNase1 was only expressed in the liver and heart of one-year-old fish but strongly expressed in the liver, spleen, gut, kidney and testis of two-year-old fish. Moreover, the immunostaining localized RNase1 production to multiple tissues of two-year-old fish. A biological functional analysis of the recombinant protein demonstrated that M. amblycephala RNase1 had a relatively strong ribonuclease activity at its optimal pH 6.1, which is consistent with the pH of its intestinal microenvironment. Collectively, these results clearly show that Ma-RNase1 protein has ribonuclease activity and the expression patterns of Ma-RNase1 are dramatically different in one year and two-year-old fish, suggesting the functional differentiation during fish growing.

Human brown adipose tissue (BAT) is recognized as one of the most important target tissues in the drug discovery for the treatment of obesity- related metabolic disorders. It is suggested that the BAT improves glucose metabolism independently of its calorigenic capacity, probably via secreting factors. Although several molecules have been identified as BAT-derived glucose metabolism-improving hormones (i.e. BATkines), the crucial factor(s) remains undiscovered. The difficulty in discovering those crucial BATkines may be attributed to the fact that Rnase1 and a variety of chymotrypsin family peptidases are expressed at relatively high levels in murine BATs, which have been used as a material in BATkine hunting. In this review, we describe a new strategy for discovering novel BATkines by using brown adipocytes (BAs) derived from human pluripotent stem cells. We also discuss the possible mechanism how human BAs are involved in the regulation of glucose metabolism.

Adaptation is one of the most fundamental issues in the studies of organismal evolution. Pancreatic ribonuclease is a very important digestive enzyme and secreted by the pancreas. Numerous studies have suggested that RNASE1 gene duplication is closely related to the functional adaptation of the digestive system in the intestinal fermentation herbivores. RNASE1 gene thus becomes one of the most important candidate genetic markers to study the molecular mechanism of adaptation of organisms to the feeding habit. Interestingly, RNASE1 gene duplication has also been found in some non-intestinal fermentation mammals, suggesting that RNASE1 gene may have produced novel tissue specificity or functions in these species. In this review, RNASE1 gene and its implications in adaptive evolution, especially in association with the feeding habit of organisms, are summarized.

Pancreatic RNase genes implicated in the adaptation of the colobine monkeys to leaf eating have long intrigued evolutionary biologists since the identification of a duplicated RNASE1 gene with enhanced digestive efficiencies in Pygathrix nemaeus. The recent emergence of two contrasting hypotheses, that is, independent duplication and one-duplication event hypotheses, make it into focus again. Current understanding of Colobine RNASE1 gene evolution of colobine monkeys largely depends on the analyses of few colobine species. The present study with more intensive taxonomic and character sampling not only provides a clearer picture of Colobine RNASE1 gene evolution but also allows to have a more thorough understanding about the molecular basis underlying the adaptation of Colobinae to the unique leaf-feeding lifestyle. The present broader and detailed phylogenetic analyses yielded two important findings: 1) All trees based on the analyses of coding, noncoding, and both regions provided consistent evidence, indicating RNASE1 duplication occurred after Asian and African colobines speciation, that is, independent duplication hypothesis; 2) No obvious evidence of gene conversion in RNASE1 gene was found, favoring independent evolution of Colobine RNASE1 gene duplicates. The conclusion drawn from previous studies that gene conversion has played a significant role in the evolution of Colobine RNASE1 was not supported. Our selective constraint analyses also provided interesting insights, with significant evidence of positive selection detected on ancestor lineages leading to duplicated gene copies. The identification of a handful of new adaptive sites and amino acid changes that have not been characterized previously also provide a necessary foundation for further experimental investigations of RNASE1 functional evolution in Colobinae.

Angiogenin (ANG) [also known as ribonuclease, RNase A family, 5 (RNASE5)], ribonuclease, RNase A family, 1 (pancreatic) (RNASE1) and ribonuclease, RNase A family, k6 (RNASE6) are three members of the RNase A superfamily. It has been suggested that these three genes play important roles in host defense. In this study, we obtained the whole open reading frame (ORF) of each gene and found the deduced proteins contain some similar structures harboring a catalytic triad and an invariant "CKXXNTF" signature motif. One single nucleotide polymorphism (SNP) was detected in each gene (g. 149GT polymorphism in the porcine ANG gene, which resulted in an amino acid change from glycine to valine, g. 296AG polymorphism in the porcine RNASE1 gene and g. 389CT polymorphism in the porcine RNASE6 gene). Association analyses revealed the significant associations (P0.05) between the porcine ANG g. 149GT polymorphism and mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean platelet volume (MPV) and platelet-large cell ratio (P-LCR) measured on 0-day-old pigs and MCV measured at 32 days after birth. The porcine RNASE6 g. 389CT polymorphism was significantly associated (P0.05) with MCV, MCH and neutrophil percentage (NEI %) measured on 0-day-old pigs, respectively. Our current findings, if confirmed by other studies, might shed some light on the roles of the investigated genes in host defense.

A morphological or physiological trait may appear multiple times in evolution. At the molecular level, similar protein functions may emerge independently in different lineages. Whether these parallel functional changes are due to parallel amino acid substitutions has been a subject of debate. Here, I address this question using digestive ribonucleases (RNases) of two groups of foregut-fermenting mammals: ruminant artiodactyls and colobine monkeys. The RNase1 gene was duplicated twice in ancestral ruminants at least 40 MYA, and it was also duplicated in the douc langur, an Asian colobine, approximately 4 MYA. After duplication, similar functional changes occurred in the ruminant and monkey enzymes. Interestingly, five amino acid substitutions in ruminant RNases that are known to affect its catalytic activity against double-stranded (ds) RNA did not occur in the monkey enzyme. Rather, a similar functional change in the monkey was caused by a different set of nine substitutions. Site-directed mutagenesis was used to make three of the five ruminant-specific substitutions in the monkey enzyme. Functional assays of these mutants showed that one of the three substitutions has a similar effect in monkeys, the second has a stronger effect, and the third has an opposite effect. These results suggest that (1) an evolutionary problem can have multiple solutions, (2) the same amino acid substitution may have opposite functional effects in homologous proteins, (3) the stochastic processes of mutation and drift play an important role even at functionally important sites, and (4) protein sequences may diverge even when their functions converge.

Although the complete genome sequences of over 50 representative species have revealed the many duplicated genes in all three domains of life1‐4, the roles of gene duplication in organismal adaptation and biodiversity are poorly understood. In addition, the evolutionary forces behind the functional divergence of duplicated genes are often unknown, leading to disagreement on the relative importance of positive Darwinian selection versus relaxation of functional constraints in this process5‐10. The methodology of earlier studies relied largely on DNA sequence analysis but lacked functional assays of duplicated genes, frequently generating contentious results 11,12 . Here we use both computational and experimental approaches to address these questions in a study of the pancreatic ribonuclease gene (RNASE1) and its duplicate gene (RNASE1B) in a leaf-eating colobine monkey, douc langur. We show that RNASE1B has evolved rapidly under positive selection for enhanced ribonucleolytic activity in an altered microenvironment, a response to increased demands for the enzyme for digesting bacterial RNA. At the same time, the ability to degrade double-stranded RNA, a non-digestive activity characteristic of primate RNASE1, has been lost in RNASE1B, indicating functional specialization and relaxation of purifying selection. Our findings demonstrate the contribution of gene duplication to organismal adaptation and show the power of combining sequence analysis and functional assays in delineating the molecular basis of adaptive evolution. A subfamily of Old World monkeys, colobines are unique primates that use leaves rather than fruits and insects as their primary food source; these leaves are then fermented by symbiotic bacteria in the foregut 13 . Similar to ruminants, colobines recover nutrients by breaking and digesting the bacteria with various enzymes, including pancreatic ribonuclease (RNASE1), which is secreted from the pancreas and transported into the small intestine to degrade RNA 14,15 . Earlier studies revealed a substantially greater amount of ribonuclease (RNase) in the pancreas of foregut fermenting mammals (colobines and ruminants) than in other mammals 14,15 . This is believed to be related to the fact that rapidly growing bacteria have the highest ratio of RNA-nitrogen to total nitrogen of all cells, and high concentrations of RNase are needed to break down bacterial RNA so that nitrogen can be recycled efficiently 14 . Using a screening method based on PCR and sequencing, we detected one RNASE1 gene in each of the 15 non-colobine primates examined, including 5 hominoids, 5 Old World monkeys, 4 New World monkeys and 1 prosimian. We determined the

Pancreatic ribonuclease (RNASE1) is a digestive enzyme that has been one of the key models in studies of evolutionary innovation and functional diversification. It has been believed that the RNASE1 gene duplications are correlated with the plant-feeding adaptation of foregut-fermenting herbivores. Here, we characterized RNASE1 genes from Caniformia, which has a simple digestive system and lacks microbial digestion typical of herbivores, in an unprecedented scope based on both gene sequence and tissue expression analyses. Remarkably, the results yielded new hypotheses regarding the evolution and the function of Caniformia RNASE1 genes. Four independent gene duplication events in the families of superfamily Musteloidea, including Procyonidae, Ailuridae, Mephitidae and Mustelidae, were recovered, rejecting previous Mustelidae-specific duplication hypothesis, but supporting Musteloidea duplication hypothesis. Moreover, our analyses revealed pronounced differences among the RNASE1 gene copies regarding their selection pressures, pI values and tissue expression patterns, suggesting the differences in their physiological functions. Notably, the expression analyses detected the transcription of a RNASE1 pseudogene in several tissues, raising the possibility that pseudogenes are also a potential source during the RNase functional diversification. In sum, the present work demonstrated a far more complex and intriguing evolutionary pattern and functional diversity of mammalian ribonuclease than previously thought.

Previous studies of protein evolution have identified important mutations in various proteins that affect a small number of residues, but dramatically alter protein function. However, the evolutionary process underlying the three-dimensional protein properties, which are determined by a much larger number of residues, remains unclear. Based on a comparative evolutionary analysis of teleost phosphoglucose isomerases (PGIs; EC 5.3.1.9), we previously demonstrated that the relatively weak selection on many amino acid sites has played an important role in the evolution of protein electric charge as a model of three-dimensional protein properties. To ascertain the generality of this finding, we sought further evidence of this type of protein evolution. For this purpose, we analyzed the vertebrate isoforms of fructose-1,6-bisphosphate aldolase (ALD; EC 4.1.2.13), for which electric charges are known to have diverged after gene duplication. The results showed that the divergence in electric charge between the ALD isoforms was also driven by weak selection on many amino acid sites, as in PGI, confirming the generality of earlier findings. To obtain further insights, ALD and PGI were compared to the proteins pancreatic ribonuclease (EC 3.1.27.5) and triose-phosphate isomerase (EC 5.3.1.1), for which electric charges likely evolved through a well-defined mode of molecular evolution; namely, strong selection on specific amino acid sites. Comparison of the number and composition of amino acids on the protein surface suggested that the absolute number of evolutionarily changeable amino acids in a protein affects the strength of selection pressure acting on individual amino acid sites.

The primary structure of pancreatic ribonuclease from langur (Presbytis entellus) has been determined. This sequence differs from that of human pancreatic ribonuclease at 14 (11%) of the amino acid positions. Eight of these 14 differences involve changes of charge, with the langur enzyme having five fewer positive charges than the human enzyme. The difference in charge between human and langur ribonuclease may be an adaptation to the different requirements for a nondigestive and a digestive role, respectively. A number of similarities in expression, gene duplications, and properties between mammalian ribonucleases and lysozymes have been observed, indicating similar adaptations in both enzyme systems.

Pancreatic ribonuclease (RNASE1) is a digestive enzyme that has been recognized to be one of the most attractive model systems for molecular evolutionary studies. The contribution of RNASE 1 gene duplication to the functional adaptation of digestive physiology in foregut-fermenting herbivores, mostly in ruminants, has been well documented. However, no one has ever done a comprehensive study on the carnivores, which are sister to the artiodactyls. Here, we sequenced this gene from 15 species of the superfamily Caniformia in order Carnivora, which all have a relatively simple digestive system and lack the microbial digestion in rumen or cecum typical of most herbivores. In contrast to our initial expectation that only a single RNASE1 gene is present in these carnivores, we observed a "birth (gene duplication)-and-death (gene deactivation)" process for the evolution of RNASE1 genes in all 3 species of Mustelidae family examined here, adding the growing diversity of RNASE1 gene family evolution. In addition, bursts of positive selection have been shown to contribute the enigmatic diversification of these RNASE1 genes in Mustelidae. The finding of the adaptive expansion of RNASE1 in animals without foregut fermentation provides another opportunity for further studies of the structure, function, and evolution of this gene, raising the possibility that new tissue specificity or other functions of RNASE 1 genes might have developed in these species.

Similar morphological or physiological changes occurring in multiple evolutionary lineages are not uncommon. Such parallel changes are believed to be adaptive, because a complex character is unlikely to originate more than once by chance. However, the occurrence of adaptive parallel amino acid substitutions is debated. Here I propose four requirements for establishing adaptive parallel evolution at the protein sequence level and use these criteria to demonstrate such a case. I report that the gene encoding pancreatic ribonuclease was duplicated independently in Asian and African leaf-eating monkeys. Statistical analyses of DNA sequences, functional assays of reconstructed ancestral proteins and site-directed mutagenesis show that the new genes acquired enhanced digestive efficiencies through parallel amino acid replacements driven by darwinian selection. They also lost a non-digestive function independently, under a relaxed selective constraint. These results demonstrate that despite the overall stochasticity, even molecular evolution has a certain degree of repeatability and predictability under the pressures of natural selection.

The physiological function of this much studied pancreatic enzyme has been misunderstood. It is essential only in ruminants and certain other herbivores, where it has a special function.