Molecular evolution of mammalian ribonucleases 1

There have been many studies on the chemistry of mammalian pancreatic ribonucleases (ribonucleases 1), but the functional biology of this family of homologous proteins is still largely unknown. Many studies have been performed on the molecular evolution and properties of this enzyme from species belonging to a large number of mammalian taxa, including paralogous gene products resulting from recent gene duplications. Novel ribonuclease 1 sequences were determined for three rodent species (gundi, brush-tailed porcupine, and squirrel), rabbit, a fruit bat, elephant, and aardvark, and the new sequences were used for deriving most parsimonious networks of ribonucleases from different mammalian orders, including earlier determined nucleotide sequences and also a larger set of protein sequences. Weak support for interordinal relationships were obtained, except for an Afrotheria clade containing elephant and aardvark. Results of current analyses and also those obtained 20 years ago on amino acid sequences confirm conclusions derived recently from larger data sets of other molecules. Several examples of recent gene duplications in ribonucleases 1 are discussed, with respect to illustrate the concepts of orthology and paralogy. Previously evidence was presented for extensive parallelism between sequence regions with attached carbohydrate (about one quarter of the molecule) of unrelated species with cecal digestion (pig and guinea pig). These features are also present in the sequences of elephant and fruit bat, species with cecal digestion, but with a very low ribonuclease content in their pancreas.     

Origin of the duplicated ribonuclease gene in guinea-pig: Comparison of the amino acid sequences with those of two close relatives: Capybara and cuis ribonuclease

The amino acid sequences of the pancreatic ribonuclease from capybara (Hydrochoerus hydrochaeris) and cuis (Galea musteloides) were determined. Both species belong to the same superfamily of the hystricomorph rodents as the guinea-pig. In guinea-pig pancreas two ribonucleases are present as a result of a recent gene duplication, but in capybara and cuis pancreas only one single ribonuclease has been found. A most parsimonious tree of ribonucleases indicates that the gene duplication leading to both guinea-pig ribonucleases occurred before the divergence of guinea-pig from capybara and cuis. This would mean that changes in expression of the ribonuclease genes have occurred in these taxa. Cuis and capybara ribonuclease have no Asn-X-Ser/Thr sequences and are carbohydrate-free proteins. Capybara ribonuclease has leucine at position 114, a position occupied by proline in the cis-configuration in bovine pancreatic ribonuclease.     

Phylogeny of ruminants secretory ribonuclease gene sequences of pronghorn (Antilocapra americana)

Phylogenetic analyses based on primary structures of mammalian ribonucleases, indicated that three homologous enzymes (pancreatic, seminal and brain ribonucleases) present in the bovine species are the results of gene duplication events, which occurred in the ancestor of the ruminants after divergence from other artiodactyls. In this paper sequences are presented of genes encoding pancreatic and brain-type ribonuclease genes of pronghorn (Antilocapra americana). The seminal-type ribonuclease gene could not be detected in this species, neither by PCR amplification nor by Southern blot analyses, indicating that it may be deleted completely in this species. Previously we demonstrated of a study of amino acid sequences of pancreatic ribonucleases of a large number of ruminants the monophyly of bovids and cervids, and that pronghorn groups with giraffe. Here we present phylogenetic analyses of nucleotide sequences of ribonucleases and other molecules from ruminant species and compare these with published data. Chevrotain (Tragulus) always groups with the other ruminants as separate taxon from the pecora or true ruminants. Within the pecora the relationships between Bovidae, Cervidae, Giraffidae, and pronghorn (Antilocapra) cannot be decided with certainty, although in the majority of analyses Antilocapra diverges first, separately or joined with giraffe. Broad taxon sampling and investigation of specific sequence features may be as important for reliable conclusions in phylogeny as the lengths of analyzed sequences.     

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

The two eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3), are among the most rapidly evolving coding sequences known among primates. The eight mouse genes identified as orthologs of EDN and ECP form a highly divergent, species-limited cluster. We present here the rat ribonuclease cluster, a group of eight distinct ribonuclease A superfamily genes that are more closely related to one another than they are to their murine counterparts. The existence of independent gene clusters suggests that numerous duplications and diversification events have occurred at these loci recently, sometime after the divergence of these two rodent species (∼10–15 million years ago). Nonsynonymous substitutions per site (d _N) calculated for the 64 mouse/rat gene pairs indicate that these ribonucleases are incorporating nonsilent mutations at accelerated rates, and comparisons of nonsynonymous to synonymous substitution (d _N / d _S) suggest that diversity in the mouse ribonuclease cluster is promoted by positive (Darwinian) selection. Although the pressures promoting similar but clearly independent styles of rapid diversification among these primate and rodent genes remain uncertain, our recent findings regarding the function of human EDN suggest a role for these ribonucleases in antiviral host defense. Received: 8 April 1999 / Accepted: 22 June 1999     

The amino-acid sequence of kangaroo pancreatic ribonuclease

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.     

Sequences related to the ox pancreatic ribonuclease coding region in the genomic DNA of mammalian species

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants.In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species.We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.Correspondence to: A. Furia     

Inclusion of Cetaceans Within the Order Artiodactyla Based on Phylogenetic Analysis of Pancreatic Ribonuclease Genes

Mammalian secretory ribonucleases (RNases 1) form a family of extensively studied homologous proteins that were already used for phylogenetic analyses at the protein sequence level previously. In this paper we report the determination of six ribonuclease gene sequences of Artiodactyla and two of Cetacea. These sequences have been used with ruminant homologues in phylogenetic analyses that supported a group including hippopotamus and toothed whales, a group of ruminant pancreatic and brain-type ribonucleases, and a group of tylopod sequences containing the Arabian camel pancreatic ribonuclease gene and Arabian and Bactrian camel and alpaca RNase 1 genes of unknown function. In all analyses the pig was the first diverging artiodactyl. This DNA-based tree is compatible to published trees derived from a number of other genes. The differences to those trees obtained with ribonuclease protein sequences can be explained by the influence of convergence of pancreatic RNases from hippopotamus, camel, and ruminants and by taking into account the information from third codon positions in the DNA-based analyses. The evolution of sequence features of ribonucleases such as the distribution of positively charged amino acids and of potential glycosylation sites is described with regard to increased double-stranded RNA cleavage that is observed in several cetacean and artiodactyl RNases which may have no role in ruminant or ruminant-like digestion. Received: 2 June 1998 / Accepted: 31 August 1998     

Primary structure of pronghorn pancreatic ribonuclease: Close relationship between giraffe and pronghorn

Summary Pancreatic ribonuclease from pronghorn (Antilocapra americana) was isolated and its amino acid sequence was determined from a tryptic digest of the performic acid-oxidized protein. Peptides were positioned by homology with other ribonucleases. Only peptides that differed in amino acid composition from the corresponding peptides of ox or goat ribonucleases were sequenced.In a most parsimonious tree of pancreatic ribonucleases, pronghorn and giraffe were placed together and these two were placed with the bovids, leaving the deer as a taxon separate from the other ruminants. The amino acid replacements that determine this tree topology are three rarely occurring replacements shared by pronghorn and giraffe. Notwithstanding their close phylogenetic relationship, both ribonucleases differ strongly in extent of glycosidation, net charge and antigenic properties.     

Carbohydrate in pancreatic ribonucleases.

A survey of the presence and compositions of carbohydrate chains attached to pancreatic ribonucleases is given. Carbohydrate chains may occur at asparagine residues in Asn-X-Ser/Thr sequences at four exposed sites of the molecule (positions 21, 34, 62 and 76). These sites form part of highly variant sequences in pancreatic ribonucleases with the consequence that the enzymes from very closely related species may differ in the presence or absence of carbohydrate. In a number of ribonucleases Asn-X-Ser/Thr sequences occur which carry carbohydrate in only a part of the molecules. The occurrence of Asn-X-Ser/Thr sequences entirely without any carbohydrate also has been demonstrated. Compositions of the carbohydrate moieties of ribonucleases from cow, sheep, pig, whales, giraffe, okapi, moose, horse, coypu, chinchilla, and guinea-pig are presented. Striking differences in complexity have been found, both between chains attached to the same site in different species (cow and giraffe), between chains attached to different sites of the same enzyme in one-species (pig) and even between chains attached to the same site in a single species (chinchilla).     

Ruminant brain ribonucleases: expression and evolution

Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving rise to three paralogous genes occurred in ruminant ancestors. One of these genes encodes a ribonuclease identified in bovine brain. A peculiar feature of this enzyme and orthologous sequences in other ruminants are C-terminal extensions consisting of 17-27 amino acid residues. Evidence was obtained by Western blot analysis for the presence of brain-type ribonucleases in brain tissue not only of ox, but also of sheep, roe deer and chevrotain (Tragulus javanicus), a member of the earliest diverged taxon of the ruminants. The C-terminal extension of brain-type ribonuclease from giraffe deviates much in sequence from orthologues in other ruminants, due to a change of reading frame. However, the gene encodes a functional enzyme, which could be expressed in heterologous systems. The messenger RNA of bovine brain ribonuclease is not only expressed at a high level in brain tissue but also in lactating mammary gland. The enzyme was isolated and identified from this latter tissue, but was not present in bovine milk, although pancreatic ribonucleases A and B could be isolated from both sources. This suggests different ways of secretion of the two enzyme types, possibly related to structural differences. The sequence of the brain-type RNase from chevrotain suggests that the C-terminal extensions of ruminant brain-type ribonucleases originate from deletions in the ancestral DNA (including a region with stop codons), followed by insertion of a 5-8-fold repeated hexanucleotide sequence, coding for a proline-rich polypeptide.     

Determination of base specificity of multiple ribonucleases from crude samples

Ribonucleases are widely found in the tissues of living organisms, but the functions of individual ribonucleases are not clear. To facilitate characterization of individual ribonucleases, I have developed a rapid method to separate and identify each ribonuclease from a crude sample by gel electrophoresis instead of by time-consuming purification steps. The ribonucleases in a crude sample are first separated by RNA-cast SDS-polyacrylamide gel electrophoresis and then eluted from the gel after ethidium bromide staining. To determine the base specificity of each ribonuclease, a 5 labelled oligonucleotide with known sequence is added to the enzyme eluate and the digested products are analyzed by denaturing gel electrophoresis. The base specificity of bovine pancreatic ribonuclease (RNase A), bullfrog oocyte-specific ribonuclease (RC-RNase), human serum ribonucleases and sweet potato leaf ribonucleases were determined by this method. Other properties of individual ribonucleases, e.g. substrate preference, may also be determined from crude samples by this method without further purification steps.Abbreviations RNase ribonuclease - SDS sodium dodecyl sulfate     

Mouse eosinophil-associated ribonucleases: a unique subfamily expressed during hematopoiesis

A unique family of ribonucleases was identified by exhaustive screening of genomic and cDNA libraries using a probe derived from a gene encoding a ribonuclease stored in the mouse eosinophil secondary granule. This family contains at least 13 genes, which encode ribonucleases, and two potential pseudogenes. The conserved sequence identity among these genes (∼70%), as well as the isolation/purification of these ribonucleases from eosinophil secondary granules, has led us to conclude that these genes form a unique clade in the mouse that we have identified as the Ear (Eosinophil-associated ribonuclease) gene family. Analyses of the nucleotide substitutions that have occurred among these ribonuclease genes reveal that duplication events within this family have been episodic, occurring within three unique periods during the past 18 × 10⁶ years. Moreover, comparisons of non-synonymous (K_a) vs. synonymous (K_s) rates of nucleotide substitution show that although these genes conserve residues necessary for RNase activity, selective evolutionary pressure(s) exist such that acquired amino acid changes appear to be advantageous. The selective advantage of these amino acid changes is currently unclear, but the occurrence of this phenomenon in both the mouse and the human highlights the importance of these changes for Ear and, therefore, eosinophil effector function(s). Received: 25 October 2000 / Accepted: 18 December 2000     

The number of nucleotides required to determine the branching order of three species,with special reference to the human-chimpanzee-gorilla divergence

Summary A mathematical theory for computing the probabilities of various nucleotide configurations among related species is developed, and the probability of obtaining the correct tree (topology) from nucleotide sequence data is evaluated using models of evolutionary trees that are close to the tree of mitochondrial DNAs from human, chimpanzee, gorilla, orangutan, and gibbon. Special attention is given to the number of nucleotides required to resolve the branching order among the three most closely related organisms (human, chimpanzee, and gorilla). If the extent of DNA divergence is close to that obtained by Brown et al. for mitochondrial DNA and if sequence data are available only for the three most closely related organisms, the number of nucleotides (m^*) required to obtain the correct tree with a probability of 95% is about 4700. If sequence data for two outgroup species (orangutan and gibbon) are available, m^* becomes about 2600–2700 when the transformed distance, distance-Wagner, maximum parsimony, or compatibility method is used. In the unweighted pair-group method, m^* is not affected by the availability of data from outgroup species. When these five different tree-making methods, as well as Fitch and Margoliash's method, are applied to the mitochondrial DNA data (1834 bp) obtained by Brown et al. and by Hixson and Brown, they all give the same phylogenetic tree, in which human and chimpanzee are most closely related. However, the trees considered here are gene trees, and to obtain the correct species tree, sequence data for several independent loci must be used.     

Magnesium ion-independent ribonucleic acid depolymerases in bacteria

The distribution of ribonucleases among bacteria has been determined from the examination of a wide variety of species. Bacteria that had been growing rapidly on a solid medium were harvested, treated with acetone and incubated in the presence of EDTA between pH4 and pH9. The ribonuclease activity was determined from the rate at which acid-soluble nucleotides were released. Out of nearly 200 strains examined, about 30 did not contain a detectable ribonuclease. The pH optima of ribonucleases in the remainder were sufficiently distinctive to suggest a use in taxonomy. Escherichia coli B was examined in more detail to determine the factors responsible for variations in the ribonuclease content of this bacterium. Growth rate had little influence on ribonuclease content when a complex medium containing no readily assimilable carbohydrate was used; the addition of glucose resulted in a marked increase in ribonuclease and a dependence of enzyme content on growth rate. An increase in the concentration of sodium chloride in the medium decreased the ribonuclease content of bacteria growing on it.     

A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences

Summary Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and, also, the evolutionary rates when the divergence times are known). In comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but both are purines or both pyrimidines, the difference is called type I (or transition type), while, if one of the two is a purine and the other is a pyrimidine, the difference is called type II (or transversion type). Letting P and Q be respectively the fractions of nucleotide sites showing type I and type II differences between two sequences compared, then the evolutionary distance per site is K = — (1/2) ln {(1 — 2P — Q) }. The evolutionary rate per year is then given by k = K/(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions per site is estimated by K'_S = — (1/2) ln (1 — 2P — Q). Also, formulae for standard errors were obtained. Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.Contribution No. 1330 from the National Institute of Genetics, Mishima, 411 Japan     

Secretory ribonuclease genes and pseudogenes in true ruminants

Mammalian pancreatic ribonucleases (RNase) form a family of extensively studied homologous proteins. Phylogenetic analyses, based on the primary structures of these enzymes, indicated that the presence of three homologous enzymes (pancreatic, seminal and brain ribonucleases) in the bovine species is due to gene duplication events, which occurred during the evolution of ancestral ruminants. In this paper the sequences are reported of the coding regions of the orthologues of the three bovine secretory ribonucleases in hog deer and roe deer, two deer species belonging to two different subfamilies of the family Cervidae. The sequences of the 3′ untranslated regions of the three different secretory RNase genes of these two deer species and giraffe are also presented. Comparison of these and previously determined sequences of ruminant ribonucleases showed that the brain-type enzymes of giraffe and these deer species exhibit variations in their C-terminal extensions. The seminal-type genes of giraffe, hog deer and roe deer show all the features of pseudogenes. Phylogenetic analyses, based on the complete coding regions and parts of the 3′ untranslated regions of the three different secretory ribonuclease genes of ox, sheep, giraffe and the two deer species, show that pancreatic, seminal- and brain-type RNases form three separate groups.     

Alkaline ribonuclease activity is increased in rat sympathetic ganglia after nerve injury

Ribonuclease activity at pH 7.1 (alkaline ribonuclease) was determined in homogenates of rat superior cervical ganglion up to 5 days after postganglionic nerve injury under optimal conditions of assay. Measurements were performed in the presence and absence of the sulfhydryl blocking agent, N-ethylmaleimide, to assess the proportion of alkaline ribonuclease apparently bound to endogenous inhibitor. Total ribonuclease activity per ganglion was stimulated 1.3 fold by 1 day after injury and remained elevated over the 5 day period. Free ribonuclease activity accounted for about 60% of the observed increase in total activity at day 1, but had returned to control level by day 3. At day 3 the entire 90% increase in total activity was attributable to ribonuclease bound to endogenous inhibitor (i.e. latent activity). These changes are occurring at times after nerve injury when marked alterations in RNA turnover have been observed, implicating alkaline ribonucleases in the control of RNA metabolism during nerve regeneration.     

Copper/Zinc superoxide dismutase: How likely is gene transfer from ponyfish toPhotobacterium leiognathi?

Summary The proposed transfer of the gene for Cu/Zn superoxide dismutase from the ponyfish to its symbiotic bacteriumPhotobacterium leiognathi has been evaluated by an extensive analysis of all available Cu/Zn superoxide dismutase sequences. By the use of four different computer programs, phylogenetic trees were constructed from the sequences of the superoxide dismutases of human, ox, pig, horse, swordfish, fruit fly, yeast, andNeurospora crassa to find out whether superoxide dismutase sequences can reliably be used for the reconstruction of genealogical relationships. All programs arrived at the same most parsimonious tree (one requiring 232 amino acid replacements), the topology of which conformed to established opinions about the phylogenetic relations among these eukaryotes, except that it placed humans closer to the artiodactyls ox and pig than it placed horses. This could be corrected at the cost of two amino acid replacements. The sequence ofP. leiognathi superoxide dismutase was then connected at all possible positions to the corrected eukaryotic tree. It was slighly more parsimonious to link the bacterial sequence to the root of the tree than to the fish branch: The former required 316 (or 317) amino acid replacements, versus 319 for the latter. This relative lack of discrimination between such distinct alternative topologies may be a general complication in the comparison of prokaryotic and eukaryotic proteins: Bacterial cytochrome c sequences also were found to be connected as parsimoniously to the root of the eukaryotic tree as to any terminal or ancestral branch. It was calculated that the rate of evolution of the bacterial superoxide dismutase gene, if transfer occurred 30 million years (Myr) ago, must have amounted to 487 amino acid replacements per 100 residues per 100 Myr. This is more than 5 times the highest rate observed in any protein (that found for fibrinopeptides), and even much higher than the maximum rate of protein evolution that can be deduced from the neutral mutation rate of unconstrained DNA. Also, no significant evidence that shared derived amino acid replacements are present in swordfish andP. leiognathi superoxide dismutase, as might be expected had gene transfer occurred, was found. On the basis of the available data it seems more reasonable to ascribe the isolated occurrence of Cu/Zn superoxide dismutase inP. leiognathi (as well as inCaulobacter crescentus) to irregular patterns of gene expression and inactivation in the course of divergent evolution than to undocumented processes of gene transfer from eukaryotes to prokaryotes.     

Allelic polymorphism in Arabian camel ribonuclease and the amino acid sequence of bactrian camel ribonuclease

Pancreatic ribonucleases from several species (whitetail deer, roe deer, guinea pig, and arabian camel) exhibit more than one amino acid at particular positions in their amino acid sequences. Since these enzymes were isolated from pooled pancreas, the origin of this heterogeneity is not clear. The pancreatic ribonucleases from 11 individual arabian camels (Camelus dromedarius) have been investigated with respect to the lysine-glutamine heterogeneity at position 103 (Welling et al., 1975). Six ribonucleases showed only one basic band and five showed two bands after polyacrylamide gel electrophoresis, suggesting a gene frequency of about 0.75 for the Lys gene and about 0.25 for the Gln gene. The amino acid sequence of bactrian camel (Camelus bactrianus) ribonuclease isolated from individual pancreatic tissue was determined and compared with that of arabian camel ribonuclease. The only difference was observed at position 103. In the ribonucleases from two unrelated bactrian camels, only glutamine was observed at that position.Part of this work has been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.).