1991), synthesized at high levels in the seminal vesicles (Furia et al. 1983), and brain ribonuclease (BRb. RNAase) (Watanabe et al. 1988). The polypeptide ...
J Mol Evol (1995) 41:850-858
jou..A MOLEEULAR o LEVOLUTION © Springer-Verlag New York Inc. 1995
Molecular Evolution of Genes Encoding Ribonucleases in Ruminant Species E. Confalone, a J.J. Beintema, 2 M.P. Sasso, 1 A. Carsana, 1 M. Palmieri, 3 M.T. Vento, 1 A. Furia 1
i Department of Organic and Biological Chemistry,UniversityFederico II of Naples, Via Mezzocannone 16, 80134 Naples, Italy 2 BiochemischLaboratorium,Universityof Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands 3 Institute of Biological Chemistry, Universityof Verona, Strada Le Grazie, 37134 Verona, Italy Received: 19 June 1995 / Accepted: 12 July 1995
Abstract. 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. Key words: Ribonuclease - - Gene duplication - Evolution - - Ruminants
Introduction
Bovine pancreatic ribonuclease (RNAase A, EC 3.1.27.5) is a well-known enzyme, secreted at a very high level by the exocrine pancreas of this species (Blackburn and Moore 1982). Homologous enzymes have also been isolated from the pancreas of other mammalian and some reptilian species (Beintema et al. 1988). Although the pancreas was the source of the enzymes in these studies, these molecules shouldn't be regarded as strictly tissue specific. Bovine RNAase A, in fact, has
Correspondence to:
A. Furia
been isolated from different organs and body fluids such as milk, urine, serum, seminal plasma, kidney, seminal vesicles, mammary glands, and salivary glands (Beintema et al. 1988). Moreover, two homologous enzymes have been isolated from ox tissues--dimeric seminal ribonuclease (BS RNAase) (Suzuki et al. 1987; D'Alessio et al. 1991), synthesized at high levels in the seminal vesicles (Furia et al. 1983), and brain ribonuclease (BRb RNAase) (Watanabe et al. 1988). The polypeptide chain of the brain enzyme is endowed with peculiar structures, such as a carboxy-terminal extension of 17 residues which is highly hydrophobic and characterized by a very high proline content and two O-linked oligosaccharide chains. The primary structures of pancreatic-type ribonucleases provided a tool for an evolutionary analysis at the molecular level. These studies indicated that the presence of three homologous enzymes in the bovine species is due to gene duplication events which occurred during the evolution of ancestral ruminants (Fitch and Beintema 1990). Later on, this conclusion was confirmed by Southern analysis of the genomic DNA of mammalian species. In fact, using as a probe the sequence encoding bovine pancreatic RNAase, a single band is generally detected in DNA isolated from nonruminant artiodactyls and other mammals, while a complex pattern of hybridization appears in genomic DNA of ruminant species (Breukelman et al. 1993). However, "brain" or "seminal" ribonucleases have been identified so far only in the bovine species. Lysozyme, another enzyme with an important digestive function in ruminants, presents very similar features to those observed for ribonucleases (Jollbs et al. 1989).
851 W e c l o n e d the genes e n c o d i n g the b o v i n e ribonucleases of the pancreas (Carsana et al. 1988) and the brain (Sasso et al. 1991) and the c D N A of the seminal e n z y m e (Palmieri et al. 1985) and, according to Southern analyses, these genes are the only ones w h i c h e n c o d e m e m b e r s of the m a m m a l i a n ribonuclease f a m i l y in the ox. A special feature o f the three b o v i n e ribonucleases is that their 3' n o n c o d i n g regions s h o w no significant seq u e n c e similarity with the 3' n o n c o d i n g regions o f two rodent ribonucleases (Schuller et al. 1990) and that they contain about 50 nucleotides repeated twice with a region rich in A and C in between. The C-terminal extension of b o v i n e brain ribonuclease is located partly in the first repeat. T h e s e features represent hot spots for gene rearrangements. H e r e i n we present data which, in a g r e e m e n t with our previous results ( B r e u k e l m a n et al. 1993), demonstrate the existence, in addition to the g e n e e n c o d i n g the ribonuclease isolated f r o m pancreatic tissues, o f genes encoding a " b r a i n " ribonuclease in giraffe (a ruminant distantly related to the ox) and sheep (belonging to the B o v i d a e like ox) revealing variations in the peculiar carb o x y - t e r m i n a l moieties of the proteins. In contrast, the " s e m i n a l " s e q u e n c e is a p s e u d o g e n e in both species. W e also report an analysis o f the transcriptional expression o f ribonuclease genes in sheep tissues. The data presented support a m o d e l for positive selection acting on the m o l e c u l a r e v o l u t i o n of ruminant ribonuclease genes.
obtained in independent reactions. Only the sequences encoding the sheep pancreas RNAase and buffalo semen RNAase were determined on clones derived from a single amplification reaction.
Purification and Analysis of RNA. Total RNA samples isolated from sheep tissues according to Chirgwin et al. (1979) were analyzed for the presence of specific transcripts by S 1 analysis. Specific probes were produced by PCR amplification on plasmid templates bearing cloned sequences according to the following scheme: oligonucleotide ABS (complementary to the region encoding aa 88-94 in sheep open reading frames [ORFs] corresponding to the pancreatic and "seminal" genes) or oligonucleotide B (complementary to the region downstream from the stop codon of the sheep "brain" ORF) was labeled with 3zp at the 5' ends and, together with oligonucleotide RP (complementary to a region close to the cloning site of the plasmid pUC19), was used as primer in PCR reaction. The thermal profile of the amplification reaction was the following: denaturing step at 94°C, annealing at 56°C, synthesis at 72°C, 30 cycles. Probe A was obtained with oligonucleotide ABS and RP as primers and a plasmid containing the ORF coding for the sheep pancreatic ribonuclease as a template. Probe B was obtained with oligonucleotide B and RP as primers and a plasmid containing the ORF encoding the "brain" ribonuclease of sheep as a template. Probe S was obtained with oligonucteotides ABS and RP as primers and a plasmid containing the sheep genomic sequence orthologous to the one encoding the bovine seminal ribonuclease as a template. The resulting probes consist of the sequence complementary to the ORF of the relative mRNA (482 nts for brain mRNA and 304 nts for pancreatic and seminal mRNAs) followed by a stretch of about 60 nucleotides, complementary to the plasmid vector. The specific activity of probes was about 2 • 1 0 6 cpm/pmol. The double-stranded fragments were fieed of precursors by ethanol precipitation in the presence of NH4OAc, annealed in formamide buffer to RNA samples at temperature 5°C lower than the T r n calculated for the mRNA-specific region of each probe, digested with S 1 endonuclease, and fractionated by PAGE in denaturing conditions.
Methods Cloning of Sequences Encoding Ribonucleases. Genomic DNA of giraffe (Giraffa camelopardalis) has been isolated from a pancreas of giraffe kindly provided by Mr. Peter Klaver of the Amsterdam Zoo "Artis"; genomic DNAs of sheep (Ovies aries), goat (Capra hircus), and buffalo (Bubalus bubalis) were kindly provided by Dr. Piero Masina, the University of Potenza, Italy. Sequences encoding ribonucleases were amplified with the Taq polymerase (Polymed or Perkin Elmer) with the hot start protocol (Ampliwax PCR Gem 100, Perkin Elmer), using as a template high-molecular-weight genomic DNA. Two different oligonucleotide pairs were used as primers in different reactions: the oligonucleotide SP (5'- GGGTCCAGCCTTCCCTGGG3'), encoding amino acids -7/-1 of bovine ribonucleases, and the oligonucleotide AS (5'-GT/cTCGGCCT/cAGGTC/AGAGA-3'), which is complementary to the region downstream from the translation termination codons of the genes encoding the pancreatic and seminal ribonucleases of the bovine species, or oligonucleotide B (5'CTTGAGTTATTGCCCTCAAGTC-3'), which is complementary to the region downstream from the stop codon of the gene encoding the bovine brain ribonuclease. The thermal profile was the following: denaturing step, the first 4 cycles at 97°C and then at 94°C, annealing at 56°C, synthesis at 72°C, 29 cycles. The amplified products were analyzed by agarose gel electrophoresis and the relevant fragments were identified by Southern analysis using the sequence encoding the bovine brain ribonuclease as a probe. The amplified products were separated by 1.2% agarose gel electrophoresis, purified, phosphorylated, treated with T4 DNA polymerase, and then cloned into the Sinai site of pUC19 or into the EcoRI site of the same plasmid after the addition of EcoRI linkers. DNA was sequenced by the dideoxynucleotide enzymatic method, using the Sequenase kit (USB). All reported sequences were determined at least twice on clones derived from amplification products
Results Amplification and Nucleotide Sequencing of Genomic Regions Coding for Pancreatic Type RibonucIeases in Some Ruminant Species The R N A a s e - s p e c i f i c amplification products p r i m e d by o l i g o n u c l e o t i d e s SP and A S consist o f f r a g m e n t s o f about 420 bp, using D N A o f giraffe or sheep as templates. The length of these fragments is identical to the one obtained with b o v i n e D N A . The R N A a s e - s p e c i f i c amplified products obtained with oligonucleotides SP and B with sheep, goat, and buffalo D N A consist of two classes of fragments o f different sizes (about 470 and 570 bp), as could also be o b s e r v e d with b o v i n e D N A (data not shown). In fact, the oligonucleotide B, w h i c h is c o m p l e m e n t a r y to the region d o w n s t r e a m f r o m the stop c o d o n o f the b o v i n e g e n e e n c o d i n g the brain ribonuclease, also hybridizes to the same gene and to the ones e n c o d i n g b o v i n e pancreatic and seminal ribonucleases in a region further downstream, yelding longer fragments. A m p l i f i c a t i o n products p r i m e d with oligonucleotides SP and B on giraffe D N A consist, on the contrary, o f a single class o f fragments of intermediate length (about 500 bp, data not shown). Several clones obtained with
852 amplification products of giraffe and sheep genomic DNA were analyzed. In each of the two species three different sequences related to the ox ribonuclease genes were found, all of them devoid of introns. We could easily assign each of these sequences as pancreatic or seminal or brain type, respectively, not only from the presence of several typical amino acid residues, but also from a most parsimonious analysis in which the three types grouped together with 100% bootstrap values. Figure 1A shows the nucleotide sequences encoding giraffe, sheep, and goat pancreatic ribonucleases compared to the coding sequence of bovine RNAase A (Carsana et al. 1988): a very high degree of identity can be observed. Only minor variations relative to the published primary structures of the proteins were found. In fact, amino acid position 76 in the giraffe sequence may be found either as Tyr (TAC) or Asn (AAC) and amino acid position 28 is Glu in our clones instead of Gln, as previously reported for the primary structure of the protein (Gaastra et al. 1974). The primary structures of sheep and goat pancreatic ribonucleases are identical, in agreement with the primary structures determined earlier by Welling et al. (1974), but different from the sheep sequence published by Kobayashi and Hirs (1973). Accordingly, the genomic sequences encoding these enzymes differ only by synonymous replacements of the goat relative to the sheep sequence. Figure 1B shows the nucleotide sequences of giraffe and sheep ORFs encoding proteins homologous to the BRb RNAase coding sequence (Sasso et al. 1991). These sequences, as well as those encoding the pancreatic enzymes, show a low proportion of nonsynonymous substitutions vs synonymous ones. In addition, the sequences shown in Fig. 1B reveal the presence of peculiar features of the protein structure which have been so far identified only in the BRb RNAase: the Arg-Arg-Arg sequence (aa positions 32-33-34) and an extension at the carboxylic end consisting of 17-19 residues. In the ox brain ribonuclease, apart from the O-glycosylation sites in the carboxy-terminal region, a single carbohydrate chain is linked to the asparagine residue 62. This N-glycosylation site is conserved and additional recognition sites have been found at asparagine residues 76 and 80 in both the giraffe and sheep "brain" ORFs. Although all the "brain" enzymes have carboxy-terminal extensions, the amino acid sequences of these regions are not conserved. This variation of protein structures may be a consequence of diverse extensive changes in lineages leading to "brain" ribonuclease genes in different ruminant species. Figure 1C shows the "seminal" nucleotide sequences of giraffe, buffalo, and sheep species compared to the sequence encoding BS RNAase (Palmieri et al. 1985; Preu~ et al. 1990). The deduced amino acid sequence of buffalo seminal RNAase is identical to the sequence of the bovine enzyme and only synonymous nucleotide substitutions are observed. A deletion occurs in DNA frag-
ments of giraffe and sheep genomes (nts 62-77 of gir-S, nt 114 of sheep-S, numbering is relative to ox-S), causing an out-of-frame joining of the downstream region and introducing stop codons at positions 83 and 128 in the giraffe and sheep sequences, respectively. In addition, in the reading frame corresponding to the one encoding the ox enzyme, many point mutations create a high proportion of nonsynonymous vs synonymous substituted codons. The histidine residue 12, which is essential for catalytic activity of mammalian ribonucleases, is replaced by leucine in the giraffe sequence or by serine in the sheep sequence. These are all typical features of pseudogenes. Some amino acid positions shared by the "seminal" sequences investigated are specific to the dimeric seminal bovine ribonuclease (Cys 32, Lys 39) or are rarely found in other ribonuclease molecules (Lys 62, Leu 28, Pro 19). Some codons of the ox sequence encoding residues unique to the BS RNAase (Cys 31, Val 102, Gly 111) are nonsynonymous codons shared by sheep and giraffe "seminal" sequences (Phe 31, Ala 102, Glu 111). It should be noted that codons for the Cys 31-Cys 32 residues, responsible for the covalent dimeric structure of the BS RNAase, are instead codons for Phe 31-Cys 32 in both the giraffe and sheep sequences:
Distribution of mRNAs Coding for Ribonucleases in Sheep Tissues The presence of specific messages in sheep pancreas, brain, seminal vesicles, mammary glands, and liver was checked by S1 analysis in order to gain insight to the specific role of each gene. Probe A, complementary to the sequence encoding the sheep pancreatic ribonuclease, was annealed to 1 ~tg of pancreas RNA or 200 gg of RNA isolated from other tissues, and the resulting pattern of protection from S 1 digestion is shown in Fig. 2A. A very strong signal corresponding to the expected size appears in the presence of RNA isolated from pancreatic tissue, while much fainter bands appear in the presence of RNA isolated from mammary glands, seminal vesicles, and brain. No signal has been detected in the presence of liver RNA. Probe B, complementary to the sequence encoding the sheep "brain" ORF, was annealed to 200 gg of RNA isolated from the five tissues examined, and the result of S 1 analysis is shown in Fig. 2B: the sheep "brain" sequence is expressed only in the central nervous system, at a fairly high level. Probe S, corresponding to the sheep genomic sequence related to the cDNA encoding the bovine seminal ribonuclease, was annealed to 200 gg of RNA isolated from the five tissues examined, and the result of S1 analysis is shown in Fig. 2C. Specific bands appear only at a long exposure time in seminal vesicles and brain, while this specific transcript is absent in mammary glands, pancreas, and liver. Very strong nonspecific signals, however, are revealed in the presence of RNA purified from sheep pan-
853
A gir-A
AAGeAATCT S C . % S C C O C C J U ~ T T ~ LysGluSezAlaAlaAlaLysgheGluArgGlnHisIleAspSerSerThrSer2~ZValSerSerSerAsnTyr IIIIII IIIIIII IIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIII III I I IIIIIIIIIIIIIIII
ox-A
LysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerSerThrSerAlaAlaSerSerSerA~nTyr IIIIII IIIIIII IIIIIIIIIIIIIIIIIIIIIIIIII sheep-A
AArOn&AT
IIIIIIIIIIII IIIIIIIIIIIIIIIIIIII
&ul-I
LysGluSezAlaAlaAlaLys~heGluArgGlnHisMetAspSerSerThrSer2M~.AlaSerSerSerAsnTyr IIIIII IIIIIII IIIIIIIII!IIIIIIIIIIIIIIII
IIIIIIIIIIII
IIIIIIIIIIIIIIIIIII
goat-A LysGluSezAlaAlaAlaLysPheGluArgGlnHisMetAspSerSerThrSerSezAlaSerSerSerAsnTyr
gir-A
ox-A
sheep-A
goat-A
gir-A
ox-A
sheep-A
goat-A
gir-A
ox-A
sheep-A
goat-A
gir-A
ox-A
sheep-A
goat-A
gir-A sheep-A goat-A
T ~ GAAC2&C~-~T~TGC.%T G A G T O 3 CysAs nGlnMetMetThzSerA/gAs nLeuThrGlnAspArgCys Lys ProVa iAsnThr PheValHi sGluSe r II III IIIIIIIII IIIIIIIII IIIIII IIIIIIIIIIIIIIII IIIIIIIIIIIIIIIII IIIIII TGTAACCAE~%T G~%CC-~-a-~-~AX~GATGCAAGCCAGTGAACAC~-~-, -x~ CC CysAs nGl nMetMet Lys SerArgAs nLeuThrLysAspArgCys Lys ProVa iAsnThr PheVa 1 Hi sGl uSer II IIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIII TGCAACCAG~GAT ~ G C A C G A G T C C CysAs nGl nMetMet Lys Se rArgAs nLeuThrGlnAspArgCys Lys ProVa iAsnThr PheVa 1 His G1 uS e r II IIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIII IIIII IIIIII TGCAACCA%GATGAT GCAAGCCAST @A ACA C ~ C~T ~~ T GAGT CC CysAsnGl nMetMetLysSe rArgAs nLeuThrGlnAspArgCys Lys ProValAsnThr PheVa 1 Hi s G1 uSe r
CTGGCTGAT _ GT GGGCAGACTAACTGCTACCASAC~ LeuAlaAspValGlnAlaValCysSerGlnLysAsnValAlaCysLysAsnGlyGlnThrAsnCysTyrGlnSer IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII II IIIIIIIIIIIII C T G G C T G A T G T C C A S G C C G T G T ~ LeuAlaAspValGlnAlaValCysSerGlnLysAsnValAlaCysLysAsnGlyGlnThrAsnCysTyrGlnSer IIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII ~ G T G C T C C C - K ~ - % ~ G T T G C C T G C T ~ LeuAlaAspValGlnAlaValCysSerGlnLysAsnValAlaCysLysAsnGlyGlnThrAsnCysTyrGlnSer IIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII CTGGO3GATGTCC~%~GO~TGT~)~-TGTTC~T~GGGC.K~%CCA~TTGCT~ LeuAlaAspValGlnAlaValCysSerGlnLysAsnValAlaCysLysP~nGlyGlnThrAsnCysTyrGlnSer
TACT CCAAST aCC~xACC~ACCJ~2C Tyr Se rAlaMet S e rl i eThrAspCysArgGluThrGl yAsnS erLysTyr ProAsnCysAl aTyrGlnThrThr IIIII IIIIIIIIIIIII IIIIIIII IIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIII IIIIIIII TACTCCACCATGAC~AT~CCAAGTACCCCAACTGTGC~TACAAGACCACC Tyr S erThrMet SerI i eThrAspCysAxgGl uThrGl ySe rSe rLysTyr ProAs nCysAl aTyrLysThrThr IIIiIIIIIIIIIIIIIIII IIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIII TACTCCACCAT CC2&AGTACCCTAACT ~ A C A A C ~ C ~ K C C TyrSe rThrMet S e r I i eThrAspCysArgGl uThrGl ySer S erLysTyr ProAs nCysAl aTyrLysThrThr IIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII ~ G C C T A C A A G A C C A C C TyrSerThrMe~SerIl eThrAspCysArgGl uThrGl ySerSerLysTyr ProAsnCysAl aTyrLysThrThr
CATTGTGGCTT ACGT~ACGATGCTTCAGTGTAG GlnAlaGlmLysHisIleIleValAlaCysGluGlyAsnProTyrValProValHisTyrAspAlaSerVal*** IIIII I IIIIIIIIIIIIIIIIIIII IIIIIIIIIII IIIIIIIIIII IIII IIIIIIIIIIIIIII ~ A A A C . % C . K T C A ~ ' i ~ T ~ ~ - ~ - r ~ A T G C T T C A G T G T A ~ GlnAlaAsnLysHisIleI!eValAlaCysGluGlyAsnProTyrValProValHisgheAspAlaSerVal*** IIIII I IIIIIIIIIII IIIIIIIIIIIIII IIIII IIIIIIIIIIIIIIIII IIIIIIIIIIIIIII ~ G G C T T ~ S G G C ~ C C C A T A ~ T G C C m E ~ ~ C A G T G T A G GlnAlaGl~LysHisIleIleValAlaCysGluGlyAsnProTyrValProValHisPheAspAlaSerVal**~ fIIII I IIIIIIIIIII IIIIIIIIIIIIII IIIII IIIII IIIIIIIIIIIIIIIIIIIIIIIIII CATAGTGGCTTGTC4%GGGGAACCCA~ACGTACCAGTCCACTTCGATGCTTCAC4~GTAG GlnAlaGluLysHisIleIleValAlaCysGluGlyAsn~roTyrValProValHisPheAspAlaSerVal***
i: 92%; i: 95.2%; i: 94.9%;
s: 15; ns: 12 s: 13; ns: 4 s: 14; ns: 4
Fig. 1. Nucleotide sequences encoding pancreatic-type ribonucleases in some ruminant species. A Sequences encoding pancreatic ribonucleases of giraffe (gir-A), ox (ox-A), sheep (sheep-A), and goat (goat-A) species. B Sequences encoding " b r a i n " ribonucleases of giraffe (girB), ox (ox-B), and sheep (sheep-B) species. C " S e m i n a l " ribonuclease O R F s of giraffe (gir-S), ox (ox-S), buffalo (bu-S), and sheep
(sheep-S) species. Predicted amino acid sequences are indicated in the three-letter code, I = nucleotide identity relative to the ox sequence, * ** = stop codon, i = percentage of nucleotide identity relative to ox sequences, s = synonymous substituted codons, ns = nonsynonymous substituted codons. Amino acid replacements relative to the ox orthologous enzymes are indicated in bold character.
854 8 qir-B
ox-8
sheep-B
A A A U m A A C T G C G G C C S C C A A G T T ~ C T ~ LysGluThEAlaAlaAlaLysPheArgArgGlnHisMer-AspSerGlySerSerSerSerSerAsnSeEAsnTyr II III IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIII ~CTGCGGCCGCCAAGTTCCC~GC~s~%~TGGACTCTGGCAGCT AC LysGluSerAlaAlaAlaLysPheArgArgGlnHisMetAspSerGlySerSerSerSerSerAsnProAsnTyr IIIIIIIIIIIIIIIIIIIIIIIIII!IIIIII!IIIIIIIIIIIIIIIIIIIIIIIIII IIIII IIIIIIII AAGGAATCTGCGGCCGCCAAGTTCC CTGGC.%GCTCCT CCAACTAC LysGluSerAlaAlaAlaLysPheArgArgGlnHisMe~AspSerGlySerSerSerSerGlyAsnSerAsnTyr
TGCAAT~GATGAAA~GT~GACACATGGACGAT~TGAA~ACCTTTGT~CC
gir-B
CysAsnGlrE4etMe~LysArgl%rgAIgMetThrHisGlyArgCysLysProValAsnThrPheValHisGluSer ox-B
sheep-B
gir-B
ox-B
sheep-B
gir-B
ox-B
sheep-B
gir-B
ox-B
sheep-B
gir-B
ox-B
sheep-B
IIIIIIIIIIIIIIIII II IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIII IIIIIIIII TGCAAT~~CGGAC~4KT~%~KC-~TG~a&C~%T~GAACACCTTTGTGCACGAGTCC CysAsnGlnMetMetLysArg/~rgArgMetThrHisGiyArgCysLysProValAsnThrPheValHisGluSer IIIII I III!IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII!IIIIIIIIIIIIIIIIIIIIIIIIIIIII TGCAACCTGATGATGAA~CGCCGGAGGATGACAC.ATGGACGAT~GAACACCTTTGTGCACGAGTCC CysAsnLeuMetMetLysArgArgArgMetThrHisGlyArgCysLys ProValAsnThrPheValHisGluSer CTGGCCGATGT~GTGCTCCCA~u&AAACJ&TCACC~GCAAC=AAT~C.AACTGCT~ LeuAlaAspValLysAlaValCysSerGlnLysAsnIleThrCysLysAsnGlyGlnProAsnCysTyrGlnSer IIII IIIIIIIIIIIIIIIIII!IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIII CTGGACGATGT~GTGCTCCD-%~J~%AC~T~~~CCCAACTGCTACCAGAC~ LeuAspAspValLysAlaValCysSerGlnLysAsnileThrCysLysAsnGlyHisProAsnCysTyrGlnSer IIIII IIIIIIIIIIIIIIIIIIIIIII!IIIIIiiIIIIIIIIIIIIIiIIIII IIII!IIIIIIIIIIIII CTGGATGATGT~TGTGCT~GCAAGhAA~C~CCCAAC~GCTAC~ LeuAspAspValLysAlaValCysSerGlnLysA~nIleThrCysLysAsnGlyGlnProAsnCysTyrGlnSer
AACT ~ G ~ . A T CACAGACT G C ~ CT~ACCCCAATT GT GC C T A F . A A C ~ A G C A.~nSerThrMetAsnI i eThrAspCysArgGl uThrGlySerSerLysTyr ProAs nCysAl aTyrLysThrSev II II IIIIIII IIIIIIIIIIII!II IIIIII!IIIIIIIlIIIIIIIIIII IIIIIIIIIIIIlIIIII AAAT CTACCAT G A C ~ . A T ~ GC ACC~CCAACTGTGCCT~ AGC LysSerThrMetSer I 1 eThrAspCysArgGluThrGlySerSerLysTyr ProAsnCysAl aTyrLysThrSe r II II IIIIIII IIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AACT C C A C C A T G A A C A T ~ C T A A G T A C C C C A A C T GTGCCTACAASACrAGC AsnSerThrMe%Asnll eThrAspCysArgGl uThrGlyGlySerLysTyr ProAsnCysAlaTyrLysThrSe r
C.~C.ACTGTGGCTTGTGAGG~a~GTATGTGCCAC~TCCACTTTGATGGTTCG GTGCTC GlnLysGlnLysTyrIleThrValAlaCysGluGlyAsnProTyrValProValHisPheAspGlySe~ValLeu IIIII!III!IIIIIIIIIIIiIIIIIIIiIIIIIIIIII! II IIIIIII!IIiIIIIIIIII IIIIII!I CA~~CACTGTGGCTTGTGAC~GCCAGTCCACTTTGAT~TGCTC GlnLysGlnLysTyr21eThrValAlaCysGluGlyAsnProTyrValProValHisPheAspGlyAlaValLeu IIIIIIIIIIIIIi II!I!III!IIIIIIIIIIIIIIIIIIII II IIIIIIIIIIIIIIIII IIIll III ~~ATATC.ACTGTGGCTTGTGAGGGAAACCCATATGTACCA~TCCACTTTGATGGTGCGGTACTC GlnLysGlnLysTyrIleThrValAlaCysGluGlyAsnProTyrValProValHisPheAspGlyAlaValLeu
TTACCTGCCACCTC~ADACAA~CCTCTGGCTC ...... ~ A A LeuProAlaThrSerThrGlnAlaGlnAlaProLettAlaA rgGly Gln~** I!IIIIIIIII II II III I I IIII I I I I TTACCTGCCACACCC~TACCCTCACTGCCACCTCCAF-ACA ...... GGCTTCTCTGA LeuProAlaThrProValProSerLeuProProProHisA rgLeuLeu*** IIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIII TTACCTGCCACACCCCTACCCTCACTGCCACCTCCAD-ACAAF~CGCAGGCTTCTCTGA LeuProAlaThrProLeuProSerLeuPro~roProHisLYsAzgArgLeuL eu**~
glr-B sneep-B
Fig. 1.
i: 90.1%; i: 96.2%;
s: 14; ns: s: 8; ns:
17 i0
Continued.
creas: these bands are likely to arise from the probe annealed to the homologous RNA encoding the pancreatic enzyme. In fact, the length of these bands closely corresponds to the positions of clusters of mismatched base pairs between sequences encoding the seminal and the pancreatic ribonucleases.
Discussion The nucleotide sequences encoding giraffe, sheep, and goat pancreatic ribonucleases (Fig. 1A) agree with the published amino acid sequences (Gaastra et al. 1974; Welling et al. 1974), with minor differences, as already
855 C AAGGAAT~T a-i~ .............. LysGluSe EAI aAl aThzLys PheGl uGlnGl n L e u M e t A s p S e r G l y ~ z S e r ProSer IIIIIIIIIIIIII IIIIIII IIII IIIII I I I I I I I I I I I I I I I I I I I I I I I I I
gir-S
ox-S LysGl uSerAl aAl aAl aLys PheGl uArgGl nHisMer-AspSe rGl yAsnSer ProSerSerSe r SerAsnTyr II Ill II IIIII II IIIII II IIIIIIIIIIII IIIIIIII IT IIIIII IIIIIIII bu-S
sheep-S
gir-S
LysGl uS erAl aAl aAl aLys PheGluArgGl nHisMetAspSerGlyAsnSer ProS er SerSer Se nAsnTyr IIIIIIIII!IIII IIIIIIIIIIIIIIIII IIII IIIIIIIII IIIIII I I I I I I I I I I I I I I I I I ~ G G C A G C T AC LysGl use rAl aAl aAl aLys PheGl uArg~isSezMet~KisSe rGlySezSer ProSerSerAsnSerAs nTyr
-- C A A C C ~ G A T G A T G T T C T ~ ~ % A C A ~ I - I - ~ - X ~
ox-S
bu-S
sheep-S
AsnLeuMetMetPheCysArgLysMetThrGlnGlyLysCys Lys ProVa iAsnThr PheGlyHisGluSe r IIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIII II IIIIIi TGCAACCTG~TGATGTGCT~GAACACCFrTGTGCATGAGTCC CysAs nLeuMetMet CysCysArgLysMet ThrGl nGl yLysCys Lys ProValAsnThr PheVa iHisGluS e r IIIIIIIIIIIIIIIIIIIIIII II IIIIIIIIIII IIIIIIII II II IIIIIIIIIII I I I I I I I I I T ~ G m ~ T G T ~ G ~ A F . A T G A G T CC CysAs nLeuMetMet CysCysArgLysMetThrGlnGlyLysCys Lys ProValAsnThr PheValHisGluSer IIIIIIIIIIIIIIII IIIIIIIIIIIIIII IIIII IIIIIIIIIIIIIIIIIIiIIIIIIIIIIIIIIIII TGCAACCTGATGAT CAC~.~S-AAATGCAAGCCA~TG~ACAECZTTGTGCATGAGT CC CysAs nLeuMetMetPheCysArgLysMetThrGl nGl y~-.ysCysLys ProVa IAsnTh r PheVa 1 HisGl uSe r
gir-S
ox-S
LeuAlaAsnValGlnAlaValCysSerGlnLysLysValIleCysLysAsnGlyLeuSerAsnCysTyrGlnSer I I I I IIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIII I IIIIIIIIIIIIIIIII IIIIII I I I I C T G G C C G A T ~ G C A A G A A T G ~ G ~ G A C ~ % A & ' ~ ~ LeuAlaAspValLysAlaValCysSerGlnLysLysValThrCysLysAsnGlyGlnThrAsnCysTyrGlnSer IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
bu-S
C
sheep-S
T
G
G
C
C
~
A
T
~
~
LeuAlaAspValLysAlaValCysSerGlnLysLysValThrCysLysAsnGlyGlnThrAsnCysTyrGlnSer I I I I I I I I I I I IIIIIIIIIIIIIIIIIIIIIIIIIII I IIIIIIIIIIIIIIII IIIIIIIIIIIIIIII C ~ G ~ ( ~ G T ~ A G G ~ C @ T ~ C G C C T ~ GCTACCAGAF~ LeuAlaAspValLysAlaValCysSerGlnLysLysValAlaCysLysAsnGlyGlnIleAsnCysTyrGlnSer
gir-S
A
ox-S
AsnSerAlaIleHisIleThrAspCysArgLysThrGlySerSerAsnTyrProAsnCysAlaTyrLysThrThr II III IIII I II IIIII IIIIII IIIIIIIIIIIII II IIIIIIIIIII IIIIIIIIIIIIIII A A A T C C A C C A T G C ~ C ~ C 4 ~ C ~ A C T G C C G C C 4 ~ A C T ~ LysSerThrMetArgIleThrAspCysArgGluThrGlySerSerLysTyrProAsnCysAlaTyrLysThrThr IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiIIIIiiiiIi
bu-S
A A A T C C A C C A T G ~ m ~ T C ~ C A S ~ ' T ~
sheep-S
LysSerThrMetArgIleThrAspCysArgGluThrGlySerSerLysTyrProAsnCysAlaTyrLysThrThr II III IIIIIIIIIII IIIIIIIIIII IIiIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AACTCCGCCATGCGCA~CGCAGACTGCCGCCAGACT ACAAGACCACC AsnSerAlaMetArgIleAlaAspCysArgGlnThrGlySerSerLysTyrProAsnCysAlaTyrLysThrThr
A
C
T
~
~
%
%
~
T
gir-S
ox-S
bu-S
sheep-S
AzgAlaGl uLysAzgl i eI 1 eValAl aCysGluGl yAsnLeu* **Va i ProVa 1 His PheAspAlaSe rVa i • ** I I7 IIIIIII IIIIIIIIIIIIIIIII II I I I II IIIIIIIIIIIIIIIIIIIIIIIIIIIIII ~ G G C T T ~ G C G G T A A A C C G T ~ G(~%L~TCGATGCTTCAGTGTAG GlnValGluLysHis Ii el leValAl aCysGl yGl yLys ProSerVal ProValHis PheAspAlaSerVal*** IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiiiiiiiiiiiiiiiiiiiiiiiiiiiii CA~GT CATAGT TAAACCGTCCGT ~ C ~ I ~ C A C T T C ~ A T G C T T C A ~ I ~ T G A GlnValGluLysHis I i eIleValAlaCysGl yGl yLys ProSerVal ProValHis PheAspAlaSerVal*** I I I IIIIIIIIIIIIIIII IIIIII!II II IIIIIII IIII IIIIIIIIIIIIIIIIIIIIIIIIII CGGGC~C/tTACTGGCTTGT~TACC4EGTCAGTCCACrTCGAT GCTT~AG ArgAIaGI uLysHisI i eI 1 ~ aCysGluGl yLys ProTyrValSerVal His PheAspAl aSerVal***
gir-S bu-S sheep-S
Fig. 1.
i: 88.6%; i: 92.8%; i: 92.8%;
s: Ii; ns: 24 s: 20; ns: 0 s: 5; ns: 18
Continued.
discussed. The data presented in this paper demonstrate the presence of ORFs coding for brain-type ribonucleases in giraffe and sheep. The complete primary structures of the putative protein products are reported. In-
variant positions so far identified in all members of the mammalian ribonucleases family (Beintema et al. 1988) are all present, suggesting that gir-B and sheep-B ORFs described in Fig. 1B belong to active genes. The car-
856 a
C
b
nt Pr
P MGSV
B
L
tR
nt Pr
540 ~ _ 504 - -
nt
Pr
L tR
P MG SV B
458 - 587 - -
434/-
P MGSV
B
2
5
L tR
540~_ ; 504458 - 434/-
540-504 - 458-434 - -
267 267 - 234-234 - -
213
213 - -
192 -184/-
192 - 184 - 1 1
2
3
4
5
6
2
3
4
5
6
7
7 1
3
4
6
7
Fig. 2. $1 analysis of sheep RNA. A Probe A digested with S1 endonuclease in the presence of 1 gg of pancreas RNA (lane 2) or 200 pg of RNA isolated from mammary glands, seminal vesicles, cerebrum, and liver (lanes 3, 4, 5, and 6, respectively). Lane 1 undigested probe, lane 7 probe digested in the presence of 200 gg of tRNA. B Probe B digested with S 1 endonuclease in the presence of 200 gg of RNA isolated from pancreas, mammary glands, seminal vesicles,
cerebrum, and liver (lanes 2-6, respectively). Lane 1 undigested probe, lane 7 probe digested in the presence of 200 gg of tRNA. C Probe S digested with S 1 endonuclease in the presence of 200 ttg of RNA isolated from pancreas, mammary glands, seminal vesicles, cerebrum, and liver (lanes 2-6, respectively). Lane 1 undigested probe, lane 7 probe digested in the presence of 200 gg of tRNA.
boxy-terminal extensions of these proteins vary greatly, as a consequence of extensive changes in the 3' region of these genomic sequences. The resulting amino acid sequences may represent neutralistic variations which are allowed as the resulting ribonuclease molecules are likely to retain the proper folding and activity. Genomic sequences of giraffe and sheep orthologous to the gene encoding the ox seminal ribonuclease are pseudogenes. On a total number of 27 RNAase-specific clones derived from ruminant DNAs with the oligonucleotide pairs SP/AS and SP/B (this work) or H1/H2 (Breukelman et al. 1993), we only identified the sequences reported in Fig. 1. Although the His 12 in the active site is replaced, most other invariant positions present in ribonucleases so far investigated are conserved (Beintema et al. 1988). The codon for Pro 19, a residue interacting with Tyr 25 and Gln 101 of the second subunit in the dimer (Capasso et al. 1983; Mazzarella et al. 1987, 1993), is also conserved in "seminal" sequences, while it is rarely found in other mammalian ribonucleases. Other residues specific to the BS RNAase such as Val 102 and Gly 111 are, instead, in both giraffe and sheep sequences, Ala 102 and Glu 111, two wellconserved amino acid positions within the ribonuclease family. Phe 31, instead, is present only in the "seminal" pseudogenes. The data presented in this work support the hypothesis that the evolutionary pathway of pancreas, brain, and semen ribonucleases might have been strictly driven by their patterns of tissue-specific expression. As a general outline, the tissue distribution of the transcripts encoding the pancreatic and cerebral ribonucleases in the sheep species parallels the one that has been observed in
ox tissues (Sasso, 1993 Ph.D. thesis, and Sasso et al., manuscript in preparation): the exocrine pancreas exclusively synthesizes the transcript encoding the pancreatic ribonuclease, although transcriptional activity of the corresponding gene is not restricted to this tissue. In both ox and sheep species ribonuclease m R N A in the central nervous system is essentially of the " b r a i n " type, A transcript corresponding to the sheep "seminal" pseudogene has also been revealed by our analyses, but neither the strict tissue specificity nor the high amount of the message, each of which has been observed in the ox (Sasso, 1993 Ph.D. thesis, and Sasso et al., manuscript in preparation), is conserved in sheep tissues. Gene duplication events may give rise to redundant expression of molecules endowed with similar properties in the same tissues. The extensive homology, which has been found in both the transcription units and flanking sequences of bovine genes encoding the cerebral and the pancreatic enzymes, suggests duplication of structural as well as regulatory regions. However, the evolution of differential gene regulatory systems (which may provide a high level of expression in the pancreas) may have favored the fixation of a duplicated gene if it supplies to a given tissue a required enzymatic activity not expressed any more by other genes of the family. Thus, the " b r a i n " RNAase genes may provide an essential ribonuclease activity in the central nervous system in which the pancreatic enzyme is not (or is very poorly) synthesized. However, further analyses are required to test this hypothesis in distantly related ruminant species. According to our results, the human gene encoding the pancreatic RNAase is transcribed in brain, pancreas, and mare-
857 mary glands (Sasso et al. manuscript in preparation) and, in addition, alkaline ribonuclease activities, sensitive to the protein inhibitor, have been detected in the CNS of many different mammalian species (Shulz-Harder and Graf v. Keyserlingk 1988), suggesting a role in brain physiology; furthermore, the human pancreatic ribonuclease has been also isolated from kidney, milk, and seminal plasma (Beintema et al. 1988). The presence of ribonuclease activities in a variety of tissues is therefore evolutionarily conserved, pointing to an important function of these enzymes in many different organs. Disulphide-linked dimeric ribonucleases have been so far isolated only from bull (D'Alessio et al. 1991) and buffalo (Farina et al. 1973) seminal vesicles and seminal plasma. At the time of gene duplication the seminal ribonuclease is a redundant function; therefore its sequence may undergo extensive variations and could even tolerate amino acid replacements unfavorable for the protein stability. One such residue may be Cys 32, which is not involved in intrachain bonds. A ribonuclease bearing Phe 31-Cys 32 residues could not fit the structure of the bovine seminal enzyme and, on the other hand, the presence of a free-SH group may hinder the stability of a monomeric enzyme. This redundant activity may be easily lost, leading to the formation of a pseudogene. On the other hand, the appearance of a stable dimeric protein (T ~ G transversion, Phe 31 --~ Cys 31 replacement), endowed with peculiar biological activities, has been fixed in the Bovidae (ox, buffalo) after divergence from the Caprinea (sheep). So here we present an example of a duplicated gene with a redundant function and low expression level during a long period of its evolutionary history. Only recently is it expressed at high level, producing a gene product with very peculiar structure and functions. The reason for the evolutionary success of the BS RNAase may reside in its ability to confer a selective advantage through its new functions. Many peculiar biological actions have been ascribed to the BS RNAase (Matousek et al. 1973a-c; Soucek et al. 1986; Tamburrini et al. 1990; Vescia et al. 1980; Laccetti et al. 1992); two of them, immunosuppression (Soucek et al. 1986; Tamburrini et al. 1990) and embryotoxic activity (Matousek et al. 1973a-c), may be relevant in the physiology of reproduction. It has been shown that impairment of T-cell mitogenic stimulation by bull seminal plasma is dependent on BS RNAase (Tamburrini et al. 1990); however, although at the present time this activity may well play a role in the reproduction of bovine species, it is conceivable that ancestral ruminants should have been endowed with immunosuppressive agents in their seminal fluids, which is a prerequisite for reproductive fitness. An intriguing hypothesis may be related to the embryotoxic activity of BS RNAase, so far demonstrated toward various rodent species. As fertilization is not affected by the presence of BS RNAase, mating of a pregnant cow with a bull carrying BS ribonuclease in its seminal plasma would lead to abortion of the previous
embryo. If a new fertilization occurs, the progeny would result BS RNAase +, provided that mating behavior of ancestral bovids had to tolerate repeated mating of pregnant cows. Such a hypothetical mechanism would favor the spreading of the BS RNAase gene in the population. To test this hypothesis, experiments are in progress in order to assess the toxic activity of BS RNAase toward bovine embryos; in addition, the structures of seminal genes and pseudogenes and of their flanking regions may help us to understand deeply the mechanism of their evolution. Acknowledgments'. We are indebted to Professor Massimo Libonati for constant support and critical reading of the manuscript. This work was partially supported by the Progetto Bilaterale of C.N.R. and a grant from the Ministero dell'Universith e della Ricerca Scientifica e Tecnologica, Italy.
References Beintema JJ, Schuller C, Irie M, Carsana A (1988) Molecular evolution of the ribonuclease superfamily. Prog Biophys Mol Biol 51:165192 Blackburn P, Moore S (1982) Pancreatic ribonuclease. In: Boyer PD (ed) The enzymes, vol 15. Academic Press, New York, 317-433 Breukelman HJ, Beintema JJ, Confalone E, Costanzo C, Sasso MP, Carsana A, Palmieri M, Furia A (1993) Sequences related to the ox pancreatic ribonuclease coding region in the genomic DNA of mammalian species, J Mol Evol 37:29-35 Capasso S, Giordano F, Mattia CA, Mazzarella L, Zagari A (1983) Refinement of the structure of bovine seminal ribonuclease. Biopolymers 22:327-332 Carsana A, Confalone E, Palmieri M, Libonati M, Furia A (1988) Structure of the bovine pancreatic ribonuclease gene: the unique intervening sequence in the 5' untranslated region contains a promoter-like element. Nucleic Acids Res 16:5491-5502 Chirgwin SM, Przybyla AE, Mac Donald RS, Putter W (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299 D'Alessio G, Di Donato A, Parente A, Piccoli R (1991) Seminal RNase: a unique member of the ribonuclease superfamily. Trends Biochem Sci 16:104-106 Farina B, Carsana A, Quesada P, De Prisco R, Leone E (1973) Ribonucleasi di vescichette seminali di bufalo. Boll Soc Ital Biol Sper 49:1450-1455 Fitch WM, Beintema JJ (1990) Correcting parsimonious trees for unseen nucleotide substitutions: the effect of dense branching as exemplified by ribonuclease. Mol Biol Evol 7:438-443 Furia A, Palmieri M, Libonati M (1983) Bovine seminal ribonuclease precursor synthesized in vitro. Biochim Biophys Acta 741:303-307 Gaastra W, Groen G, Welling GW, Beintema JJ (1974) The primary structure of giraffe pancreatic ribonuclease. FEBS Lett 41:227-233 Joll~s J, Joll~s P, Bowman BH, Prager EM, Stewart CB, Wilson AC (1989) Episodic evolution in the stomach lysozymes of ruminants. J Mol Evol 28:528-533 Kobayashi R, Hits CHW (1973) The amino acid sequence of ovine pancreatic ribonuclease A. J Biol Chem 248:7833-7837 Laccetti P, Portella G, Mastronicola MR, Russo A, Piccoli R, D'Alessio G, Vecchio G (1992) In vivo and in vitro growth-inhibitory effect of bovine seminal ribonuclease on a system of rat thyroid epithelial cells and tumors. Cancer Res 52:4582-4586 Maschhoff K, White CL, Jennings LW, Morrison-Bogorad MR (1989)
858 Ribonuclease activities and distribution in Alzheimer's and control brains. J Neurochem 52:1071-1078 Matousek J, Pavlok A, Dostal J, Grozdanovic J (1973a) Some biological properties of bull seminal vesicle aspermatogenic substance and its effect on mice. J Reprod Fertil 34:9-22 Matousek J, Fulka J, Pavlok A (1973b) Effect of ribonuclease fractions isolated from bull seminal vesicle fluid on embryonic mortality in guinea pigs, rabbits and pigs. Int J Fertil 18:13-16 Matousek J, Grozdanovic J (1973c) Specific effect of bull seminal ribonuclease (AS RNase) on cell system in mice. Comp Biochem Physiol 46A:241-248 Mazzarella L, Mattia CA, Capasso S, Di Lorenzo G (1987) Composite active sites in bovine seminal ribonuclease. Gazz Chim Ital 117: 91-97 Mazzarella L, Capasso S, Demasi D, Di Lorenzo G, Mattia CA, Zagari A (1993) Bovine Seminal ribonuclease: structure at 1.9 A resolution. Acta Crystallogr D 49:389-402. Palmieri M, Carsana A, Furia A, Libonati M (1985) Sequence analysis of a cloned cDNA coding for bovine seminal ribonuclease. Eur J Biochem 152:275-277 Preu~3 KD, Wagner S, Freudenstein J, Scheit KH (1990) Cloning of cDNA encoding the complete precursor for bovine seminal ribonuclease. Nucleic Acids Res 18:1057 Sasso MP, Carsana A, Confalone E, Cosi C, Sorrentino S, Viola M, Palmieri M, Russo E, Furia A (1991) Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain. Nucleic Acids Res 19:6469-6474
Schuller C, Nijssen HMJ, Kok R, Beintema JJ (1990) Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease. Mol Biol Evol 7:29-44 Shulz-Harder B, Graf v Keyserlingk D (1988) Comparison of brain ribonuclease of rabbit, guinea pig, rat, mouse and gerbil. Histochemistry 88:587-594 Soucek J, Chudomel V, Potmesilova I, Novak J (1986) Effect of ribonucleases on cell-mediated lympholysis reaction and on GM-CFC colonies in bone marrow culture. Nat Immun Celt Growth Regul 5:250-258 Suzuki H, Parente A, Farina B, Greco L, La Montagna R, Leone E (1987) Complete amino-acid sequence of bovine seminal ribonuclease, a dimeric protein from seminal plasma. Biol Chem Hoppe Seyler 368:1305-1312 Tamburrini M, Scala G, Verde C, Ruocco MR, Parente A, Venuta S, D'Alessio G (1990) Immunosuppressive activity of bovine seminal RNase on T-cell proliferation. Eur J Biochem 190:145-148 Vescia S, Tramontano D, Augusti-Tocco G, D'Alessio G (1980) In vitro studies on selective inhibition of tumor cell growth by seminal ribonuclease. Cancer Res 40:3740-3744 Watanabe H, Katoh H, Ishii M, Komoda Y, Sanda A, Takizawa Y, Ohgi K, Irie M (1988) Primary structure of a ribonuclease from bovine brain. J Biochem 104:939-945 Welling GW, Scheffer AJ, Beintema JJ (1974) The primary s~ucture of goat and sheep pancreatic ribonncleases. FEBS Lett 41:58~1
