﻿Mammalian ribonucleases I form a group of homologous proteins.They are pyrimidine specific and cleave RNA endonucleolyticallyto yield 3' phosphomono 
nucleotidesand oligonucleotides via 2',3' cyclic intermediates. Three homologous ribonucleases were found in ox, pancreaticRNase, seminal RNase and brain RNase. Bovine pancreaticRNase(RNaseA) consists of a single polypeptidechain of 124 anino acids, containing four disulfide bridges. Only a fraction of the secreted amount of enzyme is N-linked glycosylatedatposition34. ....

Zie: Summary...

Breukelman, Heleen Jacqueline

English

ARTS repository - University of Groningen

   University of GroningenRibonucleases 1 of ruminantsBreukelman, Heleen JacquelineIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2003Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Breukelman, H. J. (2003). Ribonucleases 1 of ruminants. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 20-06-2022l l 8 SummarySummaryMammalian ribonucleases I form a group of homologous proteins. They arepyrimidine specific and cleave RNA endonucleolytically to yield 3' phosphomononucleotides and oligonucleotides via 2',3' cyclic intermediates.Three homologous ribonucleases were found in ox, pancreatic RNase, seminalRNase and brain RNase. Bovine pancreatic RNase (RNase A) consists of a singlepolypeptide chain of 124 anino acids, containing four disulfide bridges. Only a fractionof the secreted amount of enzyme is N-linked glycosylated at position 34.Bovine seminal RNase (RNase BS) consists of two identical subunits of 124 aminoacid residues, with four disulfide bridges in each subunit. The two subunits arecovalently linked by two additional disulfide bridges between cysteine residues atpositions 3l and 32, with Cys3l of one subunit binding with Cys32 of the other subunit.No attachment sites for N-linked carbohydrate are found in the protein.Bovine brain RNase (BRb RNase) shows a high similarity to the other bovineribonucleases, except that it contains an extension of l7 amino acid resrdues at the C-terminus. This extension is proline-rich and is O-glycosylated at Thrl29 and Serl33. N-linked carbohydrate is attached at position 62.Until about l5 years ago about 40 ribonuclease sequences were determined at theprotein level. Most parsimonious analyses on these seqllences rndicated that the threedifferent bovine ribonucleases are probably products of two gene duplication events atapproximately the origin of the true ruminants, so after the divergence from the otherartiodactyls.To prove this hypothesis, investigations were started on genes coding forribonucleases in a wide variety of ruminants. Southern blot analyses of genornic DNAof various ruminants and other mammals were perfbrmed as described in Chapter 2.Several parts of the bovine pancreatic RNase gene: a fragment of the single interveningseqllence located in the 5' non-translated region, a fragment of the coding part of thegene and a fragment of the 3' non-translated region, were used as probes. Genomic DNAof most investigated mammals showed only one hybridisation band, while severalhybridisation bands coLrld be detected with ruminant genomic DNA. These resultssuggest that a unique RNase coding sequence is present in the investigirtednonruminants, whereas everal genomic regions related to the bovine pancreatic RNasegene are present in ruminants. However, recent studies show that also in othermammalian taxa gene duplications have taken place independently' 'rom the dr-rplicationsSummarJ'witli in the rur'I'o demorRNase sequeÍchain reactiondetermined. tident i ty to borespectively apr-rblished thethe three ofihrthat the 'sendeletions, whreplaced.The ribottdeer (subfamsequences ofthree dittererThe sequenctI in deer spegiraffe. Forseminal- anrThe deteboth in theRNase gencomPared rA highthese sptecshowed a ralmost dirRNase geresidues rcombine<determintypical ftMaxis9quencíSuntmarys. They arerosphomonorse, seminalof a singlely a fractionf 124 amino;ubunits areresidues atlher subunit.ther bovineres at the C-Ser133.  N-rined at theat the threern events atx the other:oding forrmic DNAChapter 2.:terveningoart of thelmic DNAle several,se resultsvestigatedtic RNasein otherplicationsSunmtar|within the ruminants.To demonstrate that the genome of giraffe possess different ribonuclease genes,RNase sequences coding for amino acids 31 to I 14 were amplified by the polymerasechain reaction and the nucleotide sequences of the cloned amplification products weredetermined. Three different RNase sequences were obtained that showed the highestidentity to bovine pancreatic RNase, bovine seminal RNase and bovine brain RNase,respectively and may code for active ribonucleases. Our colleagues in Naples meanwhilepublished the complete sequences of the three ribonuclease genes from giraffe and alsothe three orthologues fiom sheep. Most interesting of their results was the demonstrationthat the 'seminal' ribonucleases of these two species represent pseudogenes withdeletions, which disrupt the coding reading frame, in which also active site residues arereplaced.The ribonuclease genes of two deer species, hog deer (subfamiiy Cervinae) and roedeer (subfamily Odocoileinae), were investigated and the already known ribonucleaseseqllences of giraffe were extended in order to demonstrate that the existence of thethree different ribonuclease I genes extends from the bovids and giraffes to the cervids.The sequences ofthe coding regions ofthe orthologues ofthe three bovine ribonucleasesI in deer species are reported in Chapter 3 and compared with those of ox, sheep andgiraffe. For each species there are three different sequences, encoding pancreatic-,seminal- and brain-type ribonucleases.The determined pancreatic{ype RNase sequences howed a high degree of identityboth in the coding region and in the 3'untranslated region with the bovine pancreaticRNase gene. The same was observed for the obtained brain-type RNase sequencescompared with the bovine brain sequence.A high degree of identity could also be observed in the semrnal-type sequences ofthese spieces compared with the bovine seminal RNase gene. The two deer sequencesshowed a deletion or an insertion in the coding region resulting in frame shifts leadingalmost directly to a stop codon. When read in the reading frame of the bovine seminalRNase gene many non-synonymous substitutions, at positions coding for amino acidresidues essential for structure and function, could be observed. These substitutionscombined with a ratio of 2:l of non-synonymous to synonymous ubstitutions of thedetermined nucleotide sequences compared with the bovine seminal RNase gene aretypical features of pseudogenes.Maximum parsimony analyses were performed with the complete nucleotidesequences of the coding regions and the 3' untranslated regions of the pancreatic-,l 1 9seminal- and brain-type RNases of ox, sheep, giratfè, hog deer and roe cleer. Threeresulting trees showed three monophyletic groups formed by pancreatic-, seminal- andbraintype RNases, respectively, well supported by high bootstrap values. Because thetopologies in these trees of the three RNase types showed no congruency of giraffe withthe other ruminants, the sequences were also analysed by the neighbour-joining andmaximum likelihood methods. With these methods trees were obtained with the threedifferent gene-types again in separate groups, but stil l giraffe occupied non-congruentpositions relative to the bovids and cervids.In Chapter 4 the ribonuclease sequences of chevrotain, a member of the superfamilyTragulidae, the earliest diverged taxon of the ruminants, are investigated. Southern blotanalyses of chevrotain genomic DNA with a RNase I probe showed five - eighthybridisation bands. With RNase specific primers l' ive diffèrent ribonuclease Isequences could be determined by PCR amplification.Two sequences are identified as pancreatic RNase sequences, one encoding thepancreatic enzyme, the other one encoding a pseudogene. The identity of the pancreaticenzyme was confirmed by isolation of the protein and N-terminal sequence analysis. Itis the most acidic pancreatic RNase identified so far. Formation of the mature enzymerequires cleavage by signal peptidase of a peptide bond between two glutamic acidresidues. This unusual signal peptide cleavage may be due to an increase in the pK valueof the first glutamic acid, caused by the vicinity of the second residue and thehydrophobic membrane environment, resulting in an uncharged, small aliphatic residr-re,in agreement with signal peptide cleavage site requirements. The pseudogene containsthree deletions of single nucleotides at different positions in the coding sequence. Thesubstitutions in this sequence are predominantly non-synonymous and the ratio of non-synonymous to synonymous substitutions for this pseudogene compared with thepancreatic RNase gene of chevrotain is 3:1.The seminal-type RNase sequence of chevrotain shows no deletions or insertions inthe coding region and in the deduced amino acid sequence al l  catalyt ical ly act iveresidues are conserved. However, some remarkable substitr-rtions involving cysteineresidues can be observed, suggesting that also in chevrotain the seminal-type RNase maynot be expressed.The brain-type RNase sequence of chevrotain shows all the characteristics of aruminant brain-type RNase gene, like the presence of C-terminal extensions, thepresence of several putative glycosylation sites and a high excess of positive charges.The sequence possesses a deletion of the codon for amino acid 19, compared to otherSummarYbrir\nÍIof clsubstis ahno sirestdelecon\actl ÍIandbraidiveof tlgenduPevocer 'restypofmtiscpr,i nC Xfcd io iD 'c(o1 lSummaryleer. Three:minal- andiecause the3iraffe withoining andth the threel-congruent;uperfamilyuthern blotive - eightnuclease Ircoding thepancreaticanalysis. Itlre enzymetamic acidre pK valuere and thetic residue,re containsrence. Thetio of non-I with thesertions intlly activeg cysteineLl.{ase mayistics of asions, thee charges.:d to otherSumntarybrain-type ribonucleases.The fitlh sequence is a hybrid fbrmed by pancreatic and brain-type RNase sequencesof chevrotain. The part of the sequence ncoding the signal peptide has the samesubstitution in the codon for amino acid - I as the pancreatic RNase gene, and the hybridis also missing the codon for amino acid I . In the region encoding amino acids l5 - 18rro similarity with either the pancreatic or the brain-type RNase gene is observed. Therest of this hybrid is almost identical to the brain-type RNase sequence, including thedeletion of the codon for amino acid 19. Probably a gene rearrangement or geneconversion has occurred during evolution, with the region encoding amino acids l5 - 20acting as a hot spot for interchanges.Phylogenetic analyses of RNase I sequences of several ruminant, other artiodactyland whale species showed again three distinct groups of pancreatic, seminal-type andbrain-type RNases. Within each group the chevrotain sequence is the first one todiverge. Average branch length rvere used to calculate rates. In taxa with duplicationsof the RNase gene, the gene evolved at least twice as fast as in taxa in which only onegene could be observed. Probably related to new functional adaptations, the two geneduplications in ancestral ruminants caused initially an approximate fourfold increase inevolutionary rate. Later the rate slowed down again and recently diverged bovid andcervid RNases show f-ew substitutions.Brain-type ribonucleases of ruminants do not show features of pseudogenes. Theresults described in Chapter 5 indicate that the genomic sequences of ruminant brain-type RNase code for enzymatically active proteins which are expressed in brain tissueof ox, sheep, roe deer and chevrotain. The bovine enzyme is also expressed in bovinemammary gland but not in milk. However, the bovine pancreatic RNase could beisolated fiom both sources. This suggests different ways of secretion of the two enzymesprobably due to structural differences. The brain-type enzymes may be of functionalimportance in the species in which they occur with a special role of the C-terminalextensions.The most striking feature of this sequence of chevrotain brain-type RNase is an 8-fold repeat of six nucleotides with the consensus equence TCACCC, cociing for thedipeptide Ser-Pro. Alignment with the brain-type RNase sequences of bovids, deer,giraffe and pronghorn showed that this repeat occurs five times and is less wellconserved in these species. Repetitive Ser-Pro or Thr-Pro sequences are rather frequentlyobserved in protein sequences in which the hydroxy amino acids may be glycosylatedand in hinge regions between two separate folding domains in proteins. Similarl2rt22 Sunmtar y-structures may occur at the C-termini of ruminant brain-type RNases. The sequence ofthe brain-type ribonuclease of chevrotain suggests that the C-terminal extensions ofruminant brain-type RNases originate from deletions in the ancestral DNA (includinga region with stop codons), followed by insertion of a 5 to 8-fold repeatedhexanucleotide sequence, coding for a proline-rich polypeptide.The ribonuclease sequences of pronghorn, the only representative of the fourthfamily of true ruminants or pecora, are investigated in order to solve the incongruenceofthe position of giraffe. The sequences describedin Chapter ó encode the pancreaticand brain-type genes of pronghorn (Antilocapre americana). The determined brain-typesequence does not yet include a small part of the C-terminal extension and the stopcodon of this gene. In pronghorn the seminal-type ribonuclease sequence could not bedetected, neither by southern blot analysis nor by PCR amplification with primersspecific for seminal-type RNases of ruminants. This might be explained by the deletionof the complete seminal-type sequence in pronghorn and not only part of it.Maximum parsimonious analyses of nucleotide sequences of pancreatic and brain-type ribonr-rcleases of pronghorn and other ruminants with chevrotailt as outgroup wereperformed. No congruency was found between the pancreatic and brain-typeribonuclease subtrees in the obtained four most parsimonious trees. With the ma-ximumparsimony and maximum likelihood method tandemly aligned nucleotide sequences ofthe two ribonuclease types were investigated. One tree was found with the maximumparsimony method. In this tree the two bovid sequences are not monophyletic and showrather low bootstrap values. The tree obtained with the maximum likelihood methodshows giraffè and pronghom grouping together and these two again with a rnonophyleticclade of the two bovids, although the bootstrap ercentages are rather low.Phylogenetic analyses of other molecules fiom ruminant species are also presentedin this chapter. In all analyses which include chevrotain this taxon grolrps with the otherruminants as first diverging clade. Within the pecora the relationships between bovids,cervids, giraffe and pronghorn remains unresolved, although in the majority of analysespronghorn diverges first, separately or joined with giraffe. The deer species are alwaysfound to be monophyletic, br-rt the bovids are sometimes found to be diphyletic. Thismay be improved with a broader taxon sampling of bovid species, like in earlier studiesof amino acid sequences.SInrcnvatt,SamenvaRibonkornen drien hersenRNases Iribonucleopgetred(afsplitsinO m dÍu een aiDNA varin Hoofa'probe' ggenomiszoogdierbandenresultateis  in  d tgebiedeconclLlsvolgorcribonu,amino;r ibonuDeree (stdrie vin hetr iborVergvan (RNatype

Ribonucleases 1 of ruminants

University of Groningen

   University of GroningenRibonucleases 1 of ruminantsBreukelman, Heleen JacquelineIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2003Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Breukelman, H. J. (2003). Ribonucleases 1 of ruminants. Groningen: s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019l l 8 SummarySummaryMammalian ribonucleases I form a group of homologous proteins. They arepyrimidine specific and cleave RNA endonucleolytically to yield 3' phosphomononucleotides and oligonucleotides via 2',3' cyclic intermediates.Three homologous ribonucleases were found in ox, pancreatic RNase, seminalRNase and brain RNase. Bovine pancreatic RNase (RNase A) consists of a singlepolypeptide chain of 124 anino acids, containing four disulfide bridges. Only a fractionof the secreted amount of enzyme is N-linked glycosylated at position 34.Bovine seminal RNase (RNase BS) consists of two identical subunits of 124 aminoacid residues, with four disulfide bridges in each subunit. The two subunits arecovalently linked by two additional disulfide bridges between cysteine residues atpositions 3l and 32, with Cys3l of one subunit binding with Cys32 of the other subunit.No attachment sites for N-linked carbohydrate are found in the protein.Bovine brain RNase (BRb RNase) shows a high similarity to the other bovineribonucleases, except that it contains an extension of l7 amino acid resrdues at the C-terminus. This extension is proline-rich and is O-glycosylated at Thrl29 and Serl33. N-linked carbohydrate is attached at position 62.Until about l5 years ago about 40 ribonuclease sequences were determined at theprotein level. Most parsimonious analyses on these seqllences rndicated that the threedifferent bovine ribonucleases are probably products of two gene duplication events atapproximately the origin of the true ruminants, so after the divergence from the otherartiodactyls.To prove this hypothesis, investigations were started on genes coding forribonucleases in a wide variety of ruminants. Southern blot analyses of genornic DNAof various ruminants and other mammals were perfbrmed as described in Chapter 2.Several parts of the bovine pancreatic RNase gene: a fragment of the single interveningseqllence located in the 5' non-translated region, a fragment of the coding part of thegene and a fragment of the 3' non-translated region, were used as probes. Genomic DNAof most investigated mammals showed only one hybridisation band, while severalhybridisation bands coLrld be detected with ruminant genomic DNA. These resultssuggest that a unique RNase coding sequence is present in the investigirtednonruminants, whereas everal genomic regions related to the bovine pancreatic RNasegene are present in ruminants. However, recent studies show that also in othermammalian taxa gene duplications have taken place independently' 'rom the dr-rplicationsSummarJ'witli in the rur'I'o demorRNase sequeÍchain reactiondetermined. tident i ty to borespectively apr-rblished thethe three ofihrthat the 'sendeletions, whreplaced.The ribottdeer (subfamsequences ofthree dittererThe sequenctI in deer spegiraffe. Forseminal- anrThe deteboth in theRNase gencomPared rA highthese sptecshowed a ralmost dirRNase geresidues rcombine<determintypical ftMaxis9quencíSuntmarys. They arerosphomonorse, seminalof a singlely a fractionf 124 amino;ubunits areresidues atlher subunit.ther bovineres at the C-Ser133.  N-rined at theat the threern events atx the other:oding forrmic DNAChapter 2.:terveningoart of thelmic DNAle several,se resultsvestigatedtic RNasein otherplicationsSunmtar|within the ruminants.To demonstrate that the genome of giraffe possess different ribonuclease genes,RNase sequences coding for amino acids 31 to I 14 were amplified by the polymerasechain reaction and the nucleotide sequences of the cloned amplification products weredetermined. Three different RNase sequences were obtained that showed the highestidentity to bovine pancreatic RNase, bovine seminal RNase and bovine brain RNase,respectively and may code for active ribonucleases. Our colleagues in Naples meanwhilepublished the complete sequences of the three ribonuclease genes from giraffe and alsothe three orthologues fiom sheep. Most interesting of their results was the demonstrationthat the 'seminal' ribonucleases of these two species represent pseudogenes withdeletions, which disrupt the coding reading frame, in which also active site residues arereplaced.The ribonuclease genes of two deer species, hog deer (subfamiiy Cervinae) and roedeer (subfamily Odocoileinae), were investigated and the already known ribonucleaseseqllences of giraffe were extended in order to demonstrate that the existence of thethree different ribonuclease I genes extends from the bovids and giraffes to the cervids.The sequences ofthe coding regions ofthe orthologues ofthe three bovine ribonucleasesI in deer species are reported in Chapter 3 and compared with those of ox, sheep andgiraffe. For each species there are three different sequences, encoding pancreatic-,seminal- and brain-type ribonucleases.The determined pancreatic{ype RNase sequences howed a high degree of identityboth in the coding region and in the 3'untranslated region with the bovine pancreaticRNase gene. The same was observed for the obtained brain-type RNase sequencescompared with the bovine brain sequence.A high degree of identity could also be observed in the semrnal-type sequences ofthese spieces compared with the bovine seminal RNase gene. The two deer sequencesshowed a deletion or an insertion in the coding region resulting in frame shifts leadingalmost directly to a stop codon. When read in the reading frame of the bovine seminalRNase gene many non-synonymous substitutions, at positions coding for amino acidresidues essential for structure and function, could be observed. These substitutionscombined with a ratio of 2:l of non-synonymous to synonymous ubstitutions of thedetermined nucleotide sequences compared with the bovine seminal RNase gene aretypical features of pseudogenes.Maximum parsimony analyses were performed with the complete nucleotidesequences of the coding regions and the 3' untranslated regions of the pancreatic-,l 1 9seminal- and brain-type RNases of ox, sheep, giratfè, hog deer and roe cleer. Threeresulting trees showed three monophyletic groups formed by pancreatic-, seminal- andbraintype RNases, respectively, well supported by high bootstrap values. Because thetopologies in these trees of the three RNase types showed no congruency of giraffe withthe other ruminants, the sequences were also analysed by the neighbour-joining andmaximum likelihood methods. With these methods trees were obtained with the threedifferent gene-types again in separate groups, but stil l giraffe occupied non-congruentpositions relative to the bovids and cervids.In Chapter 4 the ribonuclease sequences of chevrotain, a member of the superfamilyTragulidae, the earliest diverged taxon of the ruminants, are investigated. Southern blotanalyses of chevrotain genomic DNA with a RNase I probe showed five - eighthybridisation bands. With RNase specific primers l' ive diffèrent ribonuclease Isequences could be determined by PCR amplification.Two sequences are identified as pancreatic RNase sequences, one encoding thepancreatic enzyme, the other one encoding a pseudogene. The identity of the pancreaticenzyme was confirmed by isolation of the protein and N-terminal sequence analysis. Itis the most acidic pancreatic RNase identified so far. Formation of the mature enzymerequires cleavage by signal peptidase of a peptide bond between two glutamic acidresidues. This unusual signal peptide cleavage may be due to an increase in the pK valueof the first glutamic acid, caused by the vicinity of the second residue and thehydrophobic membrane environment, resulting in an uncharged, small aliphatic residr-re,in agreement with signal peptide cleavage site requirements. The pseudogene containsthree deletions of single nucleotides at different positions in the coding sequence. Thesubstitutions in this sequence are predominantly non-synonymous and the ratio of non-synonymous to synonymous substitutions for this pseudogene compared with thepancreatic RNase gene of chevrotain is 3:1.The seminal-type RNase sequence of chevrotain shows no deletions or insertions inthe coding region and in the deduced amino acid sequence al l  catalyt ical ly act iveresidues are conserved. However, some remarkable substitr-rtions involving cysteineresidues can be observed, suggesting that also in chevrotain the seminal-type RNase maynot be expressed.The brain-type RNase sequence of chevrotain shows all the characteristics of aruminant brain-type RNase gene, like the presence of C-terminal extensions, thepresence of several putative glycosylation sites and a high excess of positive charges.The sequence possesses a deletion of the codon for amino acid 19, compared to otherSummarYbrir\nÍIof clsubstis ahno sirestdelecon\actl ÍIandbraidiveof tlgenduPevocer 'restypofmtiscpr,i nC Xfcd io iD 'c(o1 lSummaryleer. Three:minal- andiecause the3iraffe withoining andth the threel-congruent;uperfamilyuthern blotive - eightnuclease Ircoding thepancreaticanalysis. Itlre enzymetamic acidre pK valuere and thetic residue,re containsrence. Thetio of non-I with thesertions intlly activeg cysteineLl.{ase mayistics of asions, thee charges.:d to otherSumntarybrain-type ribonucleases.The fitlh sequence is a hybrid fbrmed by pancreatic and brain-type RNase sequencesof chevrotain. The part of the sequence ncoding the signal peptide has the samesubstitution in the codon for amino acid - I as the pancreatic RNase gene, and the hybridis also missing the codon for amino acid I . In the region encoding amino acids l5 - 18rro similarity with either the pancreatic or the brain-type RNase gene is observed. Therest of this hybrid is almost identical to the brain-type RNase sequence, including thedeletion of the codon for amino acid 19. Probably a gene rearrangement or geneconversion has occurred during evolution, with the region encoding amino acids l5 - 20acting as a hot spot for interchanges.Phylogenetic analyses of RNase I sequences of several ruminant, other artiodactyland whale species showed again three distinct groups of pancreatic, seminal-type andbrain-type RNases. Within each group the chevrotain sequence is the first one todiverge. Average branch length rvere used to calculate rates. In taxa with duplicationsof the RNase gene, the gene evolved at least twice as fast as in taxa in which only onegene could be observed. Probably related to new functional adaptations, the two geneduplications in ancestral ruminants caused initially an approximate fourfold increase inevolutionary rate. Later the rate slowed down again and recently diverged bovid andcervid RNases show f-ew substitutions.Brain-type ribonucleases of ruminants do not show features of pseudogenes. Theresults described in Chapter 5 indicate that the genomic sequences of ruminant brain-type RNase code for enzymatically active proteins which are expressed in brain tissueof ox, sheep, roe deer and chevrotain. The bovine enzyme is also expressed in bovinemammary gland but not in milk. However, the bovine pancreatic RNase could beisolated fiom both sources. This suggests different ways of secretion of the two enzymesprobably due to structural differences. The brain-type enzymes may be of functionalimportance in the species in which they occur with a special role of the C-terminalextensions.The most striking feature of this sequence of chevrotain brain-type RNase is an 8-fold repeat of six nucleotides with the consensus equence TCACCC, cociing for thedipeptide Ser-Pro. Alignment with the brain-type RNase sequences of bovids, deer,giraffe and pronghorn showed that this repeat occurs five times and is less wellconserved in these species. Repetitive Ser-Pro or Thr-Pro sequences are rather frequentlyobserved in protein sequences in which the hydroxy amino acids may be glycosylatedand in hinge regions between two separate folding domains in proteins. Similarl2rt22 Sunmtar y-structures may occur at the C-termini of ruminant brain-type RNases. The sequence ofthe brain-type ribonuclease of chevrotain suggests that the C-terminal extensions ofruminant brain-type RNases originate from deletions in the ancestral DNA (includinga region with stop codons), followed by insertion of a 5 to 8-fold repeatedhexanucleotide sequence, coding for a proline-rich polypeptide.The ribonuclease sequences of pronghorn, the only representative of the fourthfamily of true ruminants or pecora, are investigated in order to solve the incongruenceofthe position of giraffe. The sequences describedin Chapter ó encode the pancreaticand brain-type genes of pronghorn (Antilocapre americana). The determined brain-typesequence does not yet include a small part of the C-terminal extension and the stopcodon of this gene. In pronghorn the seminal-type ribonuclease sequence could not bedetected, neither by southern blot analysis nor by PCR amplification with primersspecific for seminal-type RNases of ruminants. This might be explained by the deletionof the complete seminal-type sequence in pronghorn and not only part of it.Maximum parsimonious analyses of nucleotide sequences of pancreatic and brain-type ribonr-rcleases of pronghorn and other ruminants with chevrotailt as outgroup wereperformed. No congruency was found between the pancreatic and brain-typeribonuclease subtrees in the obtained four most parsimonious trees. With the ma-ximumparsimony and maximum likelihood method tandemly aligned nucleotide sequences ofthe two ribonuclease types were investigated. One tree was found with the maximumparsimony method. In this tree the two bovid sequences are not monophyletic and showrather low bootstrap values. The tree obtained with the maximum likelihood methodshows giraffè and pronghom grouping together and these two again with a rnonophyleticclade of the two bovids, although the bootstrap ercentages are rather low.Phylogenetic analyses of other molecules fiom ruminant species are also presentedin this chapter. In all analyses which include chevrotain this taxon grolrps with the otherruminants as first diverging clade. Within the pecora the relationships between bovids,cervids, giraffe and pronghorn remains unresolved, although in the majority of analysespronghorn diverges first, separately or joined with giraffe. The deer species are alwaysfound to be monophyletic, br-rt the bovids are sometimes found to be diphyletic. Thismay be improved with a broader taxon sampling of bovid species, like in earlier studiesof amino acid sequences.SInrcnvatt,SamenvaRibonkornen drien hersenRNases Iribonucleopgetred(afsplitsinO m dÍu een aiDNA varin Hoofa'probe' ggenomiszoogdierbandenresultateis  in  d tgebiedeconclLlsvolgorcribonu,amino;r ibonuDeree (stdrie vin hetr iborVergvan (RNatype

BREUKELMAN, HELEEN JACQUELINE

uilis.unsyiah.ac.id

   University of GroningenRibonucleases 1 of ruminantsBreukelman, Heleen JacquelineIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2003Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Breukelman, H. J. (2003). Ribonucleases 1 of ruminants. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 05-12-2023l l 8 SummarySummaryMammalian ribonucleases I form a group of homologous proteins. They arepyrimidine specific and cleave RNA endonucleolytically to yield 3' phosphomononucleotides and oligonucleotides via 2',3' cyclic intermediates.Three homologous ribonucleases were found in ox, pancreatic RNase, seminalRNase and brain RNase. Bovine pancreatic RNase (RNase A) consists of a singlepolypeptide chain of 124 anino acids, containing four disulfide bridges. Only a fractionof the secreted amount of enzyme is N-linked glycosylated at position 34.Bovine seminal RNase (RNase BS) consists of two identical subunits of 124 aminoacid residues, with four disulfide bridges in each subunit. The two subunits arecovalently linked by two additional disulfide bridges between cysteine residues atpositions 3l and 32, with Cys3l of one subunit binding with Cys32 of the other subunit.No attachment sites for N-linked carbohydrate are found in the protein.Bovine brain RNase (BRb RNase) shows a high similarity to the other bovineribonucleases, except that it contains an extension of l7 amino acid resrdues at the C-terminus. This extension is proline-rich and is O-glycosylated at Thrl29 and Serl33. N-linked carbohydrate is attached at position 62.Until about l5 years ago about 40 ribonuclease sequences were determined at theprotein level. Most parsimonious analyses on these seqllences rndicated that the threedifferent bovine ribonucleases are probably products of two gene duplication events atapproximately the origin of the true ruminants, so after the divergence from the otherartiodactyls.To prove this hypothesis, investigations were started on genes coding forribonucleases in a wide variety of ruminants. Southern blot analyses of genornic DNAof various ruminants and other mammals were perfbrmed as described in Chapter 2.Several parts of the bovine pancreatic RNase gene: a fragment of the single interveningseqllence located in the 5' non-translated region, a fragment of the coding part of thegene and a fragment of the 3' non-translated region, were used as probes. Genomic DNAof most investigated mammals showed only one hybridisation band, while severalhybridisation bands coLrld be detected with ruminant genomic DNA. These resultssuggest that a unique RNase coding sequence is present in the investigirtednonruminants, whereas everal genomic regions related to the bovine pancreatic RNasegene are present in ruminants. However, recent studies show that also in othermammalian taxa gene duplications have taken place independently' 'rom the dr-rplicationsSummarJ'witli in the rur'I'o demorRNase sequeÍchain reactiondetermined. tident i ty to borespectively apr-rblished thethe three ofihrthat the 'sendeletions, whreplaced.The ribottdeer (subfamsequences ofthree dittererThe sequenctI in deer spegiraffe. Forseminal- anrThe deteboth in theRNase gencomPared rA highthese sptecshowed a ralmost dirRNase geresidues rcombine<determintypical ftMaxis9quencíSuntmarys. They arerosphomonorse, seminalof a singlely a fractionf 124 amino;ubunits areresidues atlher subunit.ther bovineres at the C-Ser133.  N-rined at theat the threern events atx the other:oding forrmic DNAChapter 2.:terveningoart of thelmic DNAle several,se resultsvestigatedtic RNasein otherplicationsSunmtar|within the ruminants.To demonstrate that the genome of giraffe possess different ribonuclease genes,RNase sequences coding for amino acids 31 to I 14 were amplified by the polymerasechain reaction and the nucleotide sequences of the cloned amplification products weredetermined. Three different RNase sequences were obtained that showed the highestidentity to bovine pancreatic RNase, bovine seminal RNase and bovine brain RNase,respectively and may code for active ribonucleases. Our colleagues in Naples meanwhilepublished the complete sequences of the three ribonuclease genes from giraffe and alsothe three orthologues fiom sheep. Most interesting of their results was the demonstrationthat the 'seminal' ribonucleases of these two species represent pseudogenes withdeletions, which disrupt the coding reading frame, in which also active site residues arereplaced.The ribonuclease genes of two deer species, hog deer (subfamiiy Cervinae) and roedeer (subfamily Odocoileinae), were investigated and the already known ribonucleaseseqllences of giraffe were extended in order to demonstrate that the existence of thethree different ribonuclease I genes extends from the bovids and giraffes to the cervids.The sequences ofthe coding regions ofthe orthologues ofthe three bovine ribonucleasesI in deer species are reported in Chapter 3 and compared with those of ox, sheep andgiraffe. For each species there are three different sequences, encoding pancreatic-,seminal- and brain-type ribonucleases.The determined pancreatic{ype RNase sequences howed a high degree of identityboth in the coding region and in the 3'untranslated region with the bovine pancreaticRNase gene. The same was observed for the obtained brain-type RNase sequencescompared with the bovine brain sequence.A high degree of identity could also be observed in the semrnal-type sequences ofthese spieces compared with the bovine seminal RNase gene. The two deer sequencesshowed a deletion or an insertion in the coding region resulting in frame shifts leadingalmost directly to a stop codon. When read in the reading frame of the bovine seminalRNase gene many non-synonymous substitutions, at positions coding for amino acidresidues essential for structure and function, could be observed. These substitutionscombined with a ratio of 2:l of non-synonymous to synonymous ubstitutions of thedetermined nucleotide sequences compared with the bovine seminal RNase gene aretypical features of pseudogenes.Maximum parsimony analyses were performed with the complete nucleotidesequences of the coding regions and the 3' untranslated regions of the pancreatic-,l 1 9seminal- and brain-type RNases of ox, sheep, giratfè, hog deer and roe cleer. Threeresulting trees showed three monophyletic groups formed by pancreatic-, seminal- andbraintype RNases, respectively, well supported by high bootstrap values. Because thetopologies in these trees of the three RNase types showed no congruency of giraffe withthe other ruminants, the sequences were also analysed by the neighbour-joining andmaximum likelihood methods. With these methods trees were obtained with the threedifferent gene-types again in separate groups, but stil l giraffe occupied non-congruentpositions relative to the bovids and cervids.In Chapter 4 the ribonuclease sequences of chevrotain, a member of the superfamilyTragulidae, the earliest diverged taxon of the ruminants, are investigated. Southern blotanalyses of chevrotain genomic DNA with a RNase I probe showed five - eighthybridisation bands. With RNase specific primers l' ive diffèrent ribonuclease Isequences could be determined by PCR amplification.Two sequences are identified as pancreatic RNase sequences, one encoding thepancreatic enzyme, the other one encoding a pseudogene. The identity of the pancreaticenzyme was confirmed by isolation of the protein and N-terminal sequence analysis. Itis the most acidic pancreatic RNase identified so far. Formation of the mature enzymerequires cleavage by signal peptidase of a peptide bond between two glutamic acidresidues. This unusual signal peptide cleavage may be due to an increase in the pK valueof the first glutamic acid, caused by the vicinity of the second residue and thehydrophobic membrane environment, resulting in an uncharged, small aliphatic residr-re,in agreement with signal peptide cleavage site requirements. The pseudogene containsthree deletions of single nucleotides at different positions in the coding sequence. Thesubstitutions in this sequence are predominantly non-synonymous and the ratio of non-synonymous to synonymous substitutions for this pseudogene compared with thepancreatic RNase gene of chevrotain is 3:1.The seminal-type RNase sequence of chevrotain shows no deletions or insertions inthe coding region and in the deduced amino acid sequence al l  catalyt ical ly act iveresidues are conserved. However, some remarkable substitr-rtions involving cysteineresidues can be observed, suggesting that also in chevrotain the seminal-type RNase maynot be expressed.The brain-type RNase sequence of chevrotain shows all the characteristics of aruminant brain-type RNase gene, like the presence of C-terminal extensions, thepresence of several putative glycosylation sites and a high excess of positive charges.The sequence possesses a deletion of the codon for amino acid 19, compared to otherSummarYbrir\nÍIof clsubstis ahno sirestdelecon\actl ÍIandbraidiveof tlgenduPevocer 'restypofmtiscpr,i nC Xfcd io iD 'c(o1 lSummaryleer. Three:minal- andiecause the3iraffe withoining andth the threel-congruent;uperfamilyuthern blotive - eightnuclease Ircoding thepancreaticanalysis. Itlre enzymetamic acidre pK valuere and thetic residue,re containsrence. Thetio of non-I with thesertions intlly activeg cysteineLl.{ase mayistics of asions, thee charges.:d to otherSumntarybrain-type ribonucleases.The fitlh sequence is a hybrid fbrmed by pancreatic and brain-type RNase sequencesof chevrotain. The part of the sequence ncoding the signal peptide has the samesubstitution in the codon for amino acid - I as the pancreatic RNase gene, and the hybridis also missing the codon for amino acid I . In the region encoding amino acids l5 - 18rro similarity with either the pancreatic or the brain-type RNase gene is observed. Therest of this hybrid is almost identical to the brain-type RNase sequence, including thedeletion of the codon for amino acid 19. Probably a gene rearrangement or geneconversion has occurred during evolution, with the region encoding amino acids l5 - 20acting as a hot spot for interchanges.Phylogenetic analyses of RNase I sequences of several ruminant, other artiodactyland whale species showed again three distinct groups of pancreatic, seminal-type andbrain-type RNases. Within each group the chevrotain sequence is the first one todiverge. Average branch length rvere used to calculate rates. In taxa with duplicationsof the RNase gene, the gene evolved at least twice as fast as in taxa in which only onegene could be observed. Probably related to new functional adaptations, the two geneduplications in ancestral ruminants caused initially an approximate fourfold increase inevolutionary rate. Later the rate slowed down again and recently diverged bovid andcervid RNases show f-ew substitutions.Brain-type ribonucleases of ruminants do not show features of pseudogenes. Theresults described in Chapter 5 indicate that the genomic sequences of ruminant brain-type RNase code for enzymatically active proteins which are expressed in brain tissueof ox, sheep, roe deer and chevrotain. The bovine enzyme is also expressed in bovinemammary gland but not in milk. However, the bovine pancreatic RNase could beisolated fiom both sources. This suggests different ways of secretion of the two enzymesprobably due to structural differences. The brain-type enzymes may be of functionalimportance in the species in which they occur with a special role of the C-terminalextensions.The most striking feature of this sequence of chevrotain brain-type RNase is an 8-fold repeat of six nucleotides with the consensus equence TCACCC, cociing for thedipeptide Ser-Pro. Alignment with the brain-type RNase sequences of bovids, deer,giraffe and pronghorn showed that this repeat occurs five times and is less wellconserved in these species. Repetitive Ser-Pro or Thr-Pro sequences are rather frequentlyobserved in protein sequences in which the hydroxy amino acids may be glycosylatedand in hinge regions between two separate folding domains in proteins. Similarl2rt22 Sunmtar y-structures may occur at the C-termini of ruminant brain-type RNases. The sequence ofthe brain-type ribonuclease of chevrotain suggests that the C-terminal extensions ofruminant brain-type RNases originate from deletions in the ancestral DNA (includinga region with stop codons), followed by insertion of a 5 to 8-fold repeatedhexanucleotide sequence, coding for a proline-rich polypeptide.The ribonuclease sequences of pronghorn, the only representative of the fourthfamily of true ruminants or pecora, are investigated in order to solve the incongruenceofthe position of giraffe. The sequences describedin Chapter ó encode the pancreaticand brain-type genes of pronghorn (Antilocapre americana). The determined brain-typesequence does not yet include a small part of the C-terminal extension and the stopcodon of this gene. In pronghorn the seminal-type ribonuclease sequence could not bedetected, neither by southern blot analysis nor by PCR amplification with primersspecific for seminal-type RNases of ruminants. This might be explained by the deletionof the complete seminal-type sequence in pronghorn and not only part of it.Maximum parsimonious analyses of nucleotide sequences of pancreatic and brain-type ribonr-rcleases of pronghorn and other ruminants with chevrotailt as outgroup wereperformed. No congruency was found between the pancreatic and brain-typeribonuclease subtrees in the obtained four most parsimonious trees. With the ma-ximumparsimony and maximum likelihood method tandemly aligned nucleotide sequences ofthe two ribonuclease types were investigated. One tree was found with the maximumparsimony method. In this tree the two bovid sequences are not monophyletic and showrather low bootstrap values. The tree obtained with the maximum likelihood methodshows giraffè and pronghom grouping together and these two again with a rnonophyleticclade of the two bovids, although the bootstrap ercentages are rather low.Phylogenetic analyses of other molecules fiom ruminant species are also presentedin this chapter. In all analyses which include chevrotain this taxon grolrps with the otherruminants as first diverging clade. Within the pecora the relationships between bovids,cervids, giraffe and pronghorn remains unresolved, although in the majority of analysespronghorn diverges first, separately or joined with giraffe. The deer species are alwaysfound to be monophyletic, br-rt the bovids are sometimes found to be diphyletic. Thismay be improved with a broader taxon sampling of bovid species, like in earlier studiesof amino acid sequences.SInrcnvatt,SamenvaRibonkornen drien hersenRNases Iribonucleopgetred(afsplitsinO m dÍu een aiDNA varin Hoofa'probe' ggenomiszoogdierbandenresultateis  in  d tgebiedeconclLlsvolgorcribonu,amino;r ibonuDeree (stdrie vin hetr iborVergvan (RNatype

University of Groningen Research Database

  
 University of Groningen
Ribonucleases 1 of ruminants
Breukelman, Heleen Jacqueline
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
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Publication date:
2003
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Breukelman, H. J. (2003). Ribonucleases 1 of ruminants Groningen: s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
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Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
l l 8 Summary
Summary
Mammalian ribonucleases I form a group of homologous proteins. They are
pyrimidine specific and cleave RNA endonucleolytically to yield 3' phosphomono
nucleotides and oligonucleotides via 2',3' cyclic intermediates.
Three homologous ribonucleases were found in ox, pancreatic RNase, seminal
RNase and brain RNase. Bovine pancreatic RNase (RNase A) consists of a single
polypeptide chain of 124 anino acids, containing four disulfide bridges. Only a fraction
of the secreted amount of enzyme is N-linked glycosylated at position 34.
Bovine seminal RNase (RNase BS) consists of two identical subunits of 124 amino
acid residues, with four disulfide bridges in each subunit. The two subunits are
covalently linked by two additional disulfide bridges between cysteine residues at
positions 3l and 32, with Cys3l of one subunit binding with Cys32 of the other subunit.
No attachment sites for N-linked carbohydrate are found in the protein.
Bovine brain RNase (BRb RNase) shows a high similarity to the other bovine
ribonucleases, except that it contains an extension of l7 amino acid resrdues at the C-
terminus. This extension is proline-rich and is O-glycosylated at Thrl29 and Serl33. N-
linked carbohydrate is attached at position 62.
Until about l5 years ago about 40 ribonuclease sequences were determined at the
protein level. Most parsimonious analyses on these seqllences rndicated that the three
different bovine ribonucleases are probably products of two gene duplication events at
approximately the origin of the true ruminants, so after the divergence from the other
artiodactyls.
To prove this hypothesis, investigations were started on genes coding for
ribonucleases in a wide variety of ruminants. Southern blot analyses of genornic DNA
of various ruminants and other mammals were perfbrmed as described in Chapter 2.
Several parts of the bovine pancreatic RNase gene: a fragment of the single intervening
seqllence located in the 5' non-translated region, a fragment of the coding part of the
gene and a fragment of the 3' non-translated region, were used as probes. Genomic DNA
of most investigated mammals showed only one hybridisation band, while several
hybridisation bands coLrld be detected with ruminant genomic DNA. These results
suggest that a unique RNase coding sequence is present in the investigirted
nonruminants, whereas everal genomic regions related to the bovine pancreatic RNase
gene are present in ruminants. However, recent studies show that also in other
mammalian taxa gene duplications have taken place independently' 'rom the dr-rplications
SummarJ'
witli in the rur
'I'o demor
RNase sequeÍ
chain reaction
determined. t
ident i ty to bo
respectively a
pr-rblished the
the three ofihr
that the 'sen
deletions, wh
replaced.
The ribott
deer (subfam
sequences of
three ditterer
The sequenct
I in deer spe
giraffe. For
seminal- anr
The dete
both in the
RNase gen
comPared r
A high
these sptec
showed a r
almost dir
RNase ge
residues r
combine<
determin
typical ft
Maxi
s9quencí
Suntmary
s. They are
rosphomono
rse, seminal
of a single
ly a fraction
f 124 amino
;ubunits are
residues at
lher subunit.
ther bovine
res at the C-
Ser133.  N-
rined at the
at the three
rn events at
x the other
:oding for
rmic DNA
Chapter 2.
:tervening
oart of the
lmic DNA
le several
,se results
vestigated
tic RNase
in other
plications
Sunmtar|
within the ruminants.
To demonstrate that the genome of giraffe possess different ribonuclease genes,
RNase sequences coding for amino acids 31 to I 14 were amplified by the polymerase
chain reaction and the nucleotide sequences of the cloned amplification products were
determined. Three different RNase sequences were obtained that showed the highest
identity to bovine pancreatic RNase, bovine seminal RNase and bovine brain RNase,
respectively and may code for active ribonucleases. Our colleagues in Naples meanwhile
published the complete sequences of the three ribonuclease genes from giraffe and also
the three orthologues fiom sheep. Most interesting of their results was the demonstration
that the 'seminal' ribonucleases of these two species represent pseudogenes with
deletions, which disrupt the coding reading frame, in which also active site residues are
replaced.
The ribonuclease genes of two deer species, hog deer (subfamiiy Cervinae) and roe
deer (subfamily Odocoileinae), were investigated and the already known ribonuclease
seqllences of giraffe were extended in order to demonstrate that the existence of the
three different ribonuclease I genes extends from the bovids and giraffes to the cervids.
The sequences ofthe coding regions ofthe orthologues ofthe three bovine ribonucleases
I in deer species are reported in Chapter 3 and compared with those of ox, sheep and
giraffe. For each species there are three different sequences, encoding pancreatic-,
seminal- and brain-type ribonucleases.
The determined pancreatic{ype RNase sequences howed a high degree of identity
both in the coding region and in the 3'untranslated region with the bovine pancreatic
RNase gene. The same was observed for the obtained brain-type RNase sequences
compared with the bovine brain sequence.
A high degree of identity could also be observed in the semrnal-type sequences of
these spieces compared with the bovine seminal RNase gene. The two deer sequences
showed a deletion or an insertion in the coding region resulting in frame shifts leading
almost directly to a stop codon. When read in the reading frame of the bovine seminal
RNase gene many non-synonymous substitutions, at positions coding for amino acid
residues essential for structure and function, could be observed. These substitutions
combined with a ratio of 2:l of non-synonymous to synonymous ubstitutions of the
determined nucleotide sequences compared with the bovine seminal RNase gene are
typical features of pseudogenes.
Maximum parsimony analyses were performed with the complete nucleotide
sequences of the coding regions and the 3' untranslated regions of the pancreatic-,
l 1 9
seminal- and brain-type RNases of ox, sheep, giratfè, hog deer and roe cleer. Three
resulting trees showed three monophyletic groups formed by pancreatic-, seminal- and
braintype RNases, respectively, well supported by high bootstrap values. Because the
topologies in these trees of the three RNase types showed no congruency of giraffe with
the other ruminants, the sequences were also analysed by the neighbour-joining and
maximum likelihood methods. With these methods trees were obtained with the three
different gene-types again in separate groups, but stil l giraffe occupied non-congruent
positions relative to the bovids and cervids.
In Chapter 4 the ribonuclease sequences of chevrotain, a member of the superfamily
Tragulidae, the earliest diverged taxon of the ruminants, are investigated. Southern blot
analyses of chevrotain genomic DNA with a RNase I probe showed five - eight
hybridisation bands. With RNase specific primers l' ive diffèrent ribonuclease I
sequences could be determined by PCR amplification.
Two sequences are identified as pancreatic RNase sequences, one encoding the
pancreatic enzyme, the other one encoding a pseudogene. The identity of the pancreatic
enzyme was confirmed by isolation of the protein and N-terminal sequence analysis. It
is the most acidic pancreatic RNase identified so far. Formation of the mature enzyme
requires cleavage by signal peptidase of a peptide bond between two glutamic acid
residues. This unusual signal peptide cleavage may be due to an increase in the pK value
of the first glutamic acid, caused by the vicinity of the second residue and the
hydrophobic membrane environment, resulting in an uncharged, small aliphatic residr-re,
in agreement with signal peptide cleavage site requirements. The pseudogene contains
three deletions of single nucleotides at different positions in the coding sequence. The
substitutions in this sequence are predominantly non-synonymous and the ratio of non-
synonymous to synonymous substitutions for this pseudogene compared with the
pancreatic RNase gene of chevrotain is 3:1.
The seminal-type RNase sequence of chevrotain shows no deletions or insertions in
the coding region and in the deduced amino acid sequence al l  catalyt ical ly act ive
residues are conserved. However, some remarkable substitr-rtions involving cysteine
residues can be observed, suggesting that also in chevrotain the seminal-type RNase may
not be expressed.
The brain-type RNase sequence of chevrotain shows all the characteristics of a
ruminant brain-type RNase gene, like the presence of C-terminal extensions, the
presence of several putative glycosylation sites and a high excess of positive charges.
The sequence possesses a deletion of the codon for amino acid 19, compared to other
SummarY
brir\n
Í
I
of cl
subst
is ah
no si
rest
dele
con\
actl Í
I
and
brai
dive
of tl
gen
duP
evo
cer '
res
typ
of
mt
isc
pr,
i n
C X
fc
d i
o i
D '
c(
o
1 l
Summary
leer. Three
:minal- and
iecause the
3iraffe with
oining and
th the three
l-congruent
;uperfamily
uthern blot
ive - eight
nuclease I
rcoding the
pancreatic
analysis. It
lre enzyme
tamic acid
re pK value
re and the
tic residue,
re contains
rence. The
tio of non-
I with the
sertions in
tlly active
g cysteine
Ll.{ase may
istics of a
sions, the
e charges.
:d to other
Sumntary
brain-type ribonucleases.
The fitlh sequence is a hybrid fbrmed by pancreatic and brain-type RNase sequences
of chevrotain. The part of the sequence ncoding the signal peptide has the same
substitution in the codon for amino acid - I as the pancreatic RNase gene, and the hybrid
is also missing the codon for amino acid I . In the region encoding amino acids l5 - 18
rro similarity with either the pancreatic or the brain-type RNase gene is observed. The
rest of this hybrid is almost identical to the brain-type RNase sequence, including the
deletion of the codon for amino acid 19. Probably a gene rearrangement or gene
conversion has occurred during evolution, with the region encoding amino acids l5 - 20
acting as a hot spot for interchanges.
Phylogenetic analyses of RNase I sequences of several ruminant, other artiodactyl
and whale species showed again three distinct groups of pancreatic, seminal-type and
brain-type RNases. Within each group the chevrotain sequence is the first one to
diverge. Average branch length rvere used to calculate rates. In taxa with duplications
of the RNase gene, the gene evolved at least twice as fast as in taxa in which only one
gene could be observed. Probably related to new functional adaptations, the two gene
duplications in ancestral ruminants caused initially an approximate fourfold increase in
evolutionary rate. Later the rate slowed down again and recently diverged bovid and
cervid RNases show f-ew substitutions.
Brain-type ribonucleases of ruminants do not show features of pseudogenes. The
results described in Chapter 5 indicate that the genomic sequences of ruminant brain-
type RNase code for enzymatically active proteins which are expressed in brain tissue
of ox, sheep, roe deer and chevrotain. The bovine enzyme is also expressed in bovine
mammary gland but not in milk. However, the bovine pancreatic RNase could be
isolated fiom both sources. This suggests different ways of secretion of the two enzymes
probably due to structural differences. The brain-type enzymes may be of functional
importance in the species in which they occur with a special role of the C-terminal
extensions.
The most striking feature of this sequence of chevrotain brain-type RNase is an 8-
fold repeat of six nucleotides with the consensus equence TCACCC, cociing for the
dipeptide Ser-Pro. Alignment with the brain-type RNase sequences of bovids, deer,
giraffe and pronghorn showed that this repeat occurs five times and is less well
conserved in these species. Repetitive Ser-Pro or Thr-Pro sequences are rather frequently
observed in protein sequences in which the hydroxy amino acids may be glycosylated
and in hinge regions between two separate folding domains in proteins. Similar
l2r
t22 Sunmtar y-
structures may occur at the C-termini of ruminant brain-type RNases. The sequence of
the brain-type ribonuclease of chevrotain suggests that the C-terminal extensions of
ruminant brain-type RNases originate from deletions in the ancestral DNA (including
a region with stop codons), followed by insertion of a 5 to 8-fold repeated
hexanucleotide sequence, coding for a proline-rich polypeptide.
The ribonuclease sequences of pronghorn, the only representative of the fourth
family of true ruminants or pecora, are investigated in order to solve the incongruence
ofthe position of giraffe. The sequences describedin Chapter ó encode the pancreatic
and brain-type genes of pronghorn (Antilocapre americana). The determined brain-type
sequence does not yet include a small part of the C-terminal extension and the stop
codon of this gene. In pronghorn the seminal-type ribonuclease sequence could not be
detected, neither by southern blot analysis nor by PCR amplification with primers
specific for seminal-type RNases of ruminants. This might be explained by the deletion
of the complete seminal-type sequence in pronghorn and not only part of it.
Maximum parsimonious analyses of nucleotide sequences of pancreatic and brain-
type ribonr-rcleases of pronghorn and other ruminants with chevrotailt as outgroup were
performed. No congruency was found between the pancreatic and brain-type
ribonuclease subtrees in the obtained four most parsimonious trees. With the ma-ximum
parsimony and maximum likelihood method tandemly aligned nucleotide sequences of
the two ribonuclease types were investigated. One tree was found with the maximum
parsimony method. In this tree the two bovid sequences are not monophyletic and show
rather low bootstrap values. The tree obtained with the maximum likelihood method
shows giraffè and pronghom grouping together and these two again with a rnonophyletic
clade of the two bovids, although the bootstrap ercentages are rather low.
Phylogenetic analyses of other molecules fiom ruminant species are also presented
in this chapter. In all analyses which include chevrotain this taxon grolrps with the other
ruminants as first diverging clade. Within the pecora the relationships between bovids,
cervids, giraffe and pronghorn remains unresolved, although in the majority of analyses
pronghorn diverges first, separately or joined with giraffe. The deer species are always
found to be monophyletic, br-rt the bovids are sometimes found to be diphyletic. This
may be improved with a broader taxon sampling of bovid species, like in earlier studies
of amino acid sequences.
SInrcnvatt,
Samenva
Ribon
kornen dri
en hersen
RNases I
ribonucle
opgetred(
afsplitsin
O m d
Íu een ai
DNA var
in Hoofa
'probe' g
genomis
zoogdier
banden
resultate
is  in  d t
gebiede
conclLls
volgorc
ribonu,
amino;
r ibonu
De
ree (st
drie v
in het
r ibor
Verg
van (
RNa
type


Ribonucleases 1 of ruminants

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