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.

The differential pattern of tissue-specific expression of ruminant pancreatic type ribonucleases may help to understand the evolutionary history of their genes.

Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving three paralogous genes occurred in ruminant ancestors. The enzymes of the bovine species encoded by these genes, isolated from pancreas, brain and seminal vesicles, present similar enzymological properties but distinct structural features. In other ruminant species, genomic sequences orthologous to the bovine genes of pancreas and brain ribonucleases encode active enzymes. In mammalian species other than ruminant artiodactyls, only one gene encoding ribonuclease of the pancreatic type is generally present. In this work, we describe a differential pattern of transcriptional expression of the pancreas and brain ribonuclease genes in the ox species and report transcription of the human ribonuclease gene in brain as well as in pancreas and in mammary gland. We also report the molecular cloning of the gene encoding the bovine seminal ribonuclease in which the structural organization already described for the two paralogous genes is conserved. The seminal RNAase is exclusively expressed in seminal vesicles of Bos taurus, whereas in other ruminant species, the orthologous sequence is a pseudogene. Previous studies from a number of research groups demonstrated that, unlike other mammalian ribonucleases, the seminal enzyme is a covalent dimer, and its unique quaternary structure correlates with special biological activities. The major determinant of dimer formation, i.e. the presence of two adjacent cysteine residues, is absent in the pseudogenes. We advance the hypothesis that the differentiation of distinct expression patterns could represent an important evolutionary determinant for the genes encoding pancreas and brain ribonucleases in ruminants, whereas the differentiation of a quaternary structure endowed with new biological functions could be the main determinant for the evolutionary success of the seminal gene in the bovine species.

Molecular evolution of genes encoding ribonucleases in ruminant species.

Phylogenetic analysis, based on the primary structures of mammalian pancreatic-type ribonucleases, indicated that gene duplication events, which occurred during the evolution of ancestral ruminants, gave rise to the three paralogous enzymes present in the bovine species. Herein we report data that demonstrate the existence of the orthologues of the bovine pancreatic, seminal, and cerebral ribonucleases coding sequences in the genomes of giraffe and sheep. The "seminal" sequence is a pseudogene in both species. We also report an analysis of the transcriptional expression of ribonuclease genes in sheep tissues. The data presented support a model for positive selection acting on the molecular evolution of ruminant ribonuclease genes.

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.

Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain.

In this paper we report the molecular cloning of the gene encoding the bovine brain ribonuclease. The nucleotide sequence determined in this work shows a high degree of identity to the homologous gene encoding the bovine pancreatic ribonuclease. Processing of the primary transcripts of these genes also follows a similar pathway, splicing of the unique intron in the 5' untranslated region occurs at corresponding positions. Expression of the bovine brain ribonuclease gene can be detected both at the transcriptional and translational levels in all the regions of the brain examined.

Structure of the bovine pancreatic ribonuclease gene: the unique intervening sequence in the 5' untranslated region contains a promoter-like element.

Although pancreatic ribonucleases are extensively studied proteins, little information is available on nucleic acids coding for these enzymes. Here, for the first time, the structure of a gene coding for such an enzyme, the well known bovine pancreatic ribonuclease, is reported. The coding region of this gene is devoid of introns, whereas the 5' untranslated sequence of the pancreatic transcript contains an intron of 735 nucleotides. This intervening sequence is endowed with signals (CAAT and TATA boxes) which might act as regulatory elements. The structural organization of this gene suggests that the sequence coding for the bovine pancreatic ribonuclease might be expressed under the control of two different promoters.