the eutherian kallikrein genes were implicated in major physiological and pathological processes , as well as in medical molecular diagnostics , , , . for example , the serum level of human prostate - specific antigen abundant in seminal plasma was used as molecular marker in diagnostics of prostate cancer . the comprehensive eutherian kallikrein gene data sets established major criteria of kallikrein gene annotations , including genomic localization within single genomic kallikrein locus region , common kallikrein gene features including 5 translated exons , as well as patterns of kallikrein protein amino acid sequence similarities including invariant catalytic triad amino acid sites his , asp and ser and distribution of common cysteine amino acid residues , , , , , , , , , , . specifically , the human kallikreins comprised 15 serine peptidases , including 3 classical kallikreins klk1 or paradigmatic pancreatic kallikrein , klk2 or glandular kallikrein and klk3 or prostate - specific antigen , as well as klk4klk15 kallikrein - related serine peptidases . nevertheless , future updates and revisions of comprehensive eutherian gene data sets were expected , due to the incompleteness of public eutherian genomic sequence data sets ,   and potential genomic sequence errors . for example , the so - called lexicographical bias was described in some genomic sequence assembly programs   and phylogenetic analyses could be affected by potential genomic sequence errors . thus , the eutherian comparative genomic analysis protocol applicable in curation of major eutherian gene data sets was established as assistance in protection against potential genomic sequence errors , , . the protocol integrated gene annotations , phylogenetic analysis and protein molecular evolution analysis with new genomics and protein molecular evolution tests into one framework of eutherian gene descriptions . for example , the protocol revised , updated and published 9 major eutherian gene data sets , including 1172 complete coding sequences deposited in european nucleotide archive as curated third party data gene data sets . using eutherian comparative genomic analysis protocol and public genomic sequence assemblies , , , the present study made attempts to update and revise eutherian kallikrein kln genes . the sequence alignment editor bioedit 7.0.5.3 was used in analyses and manipulations of kln nucleotide and protein sequences ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) . the public eutherian genomic sequence assemblies used in gene identifications of potential kln coding sequences were downloaded from national center for biotechnology information ( ncbi ) genbank ( ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/vertebrate_mammalian/ ) , as well as ncbi 's blast programs ( ftp://ftp.ncbi.nlm.nih.gov/blast/ ) . in addition , the public eutherian genomic sequences were downloaded from ensembl genome browser ( http://www.ensembl.org ) . the analyses of kln gene features included direct evidence of gene annotations deposited in ncbi 's nr , est_human , est_mouse and est_others databases ( https://www.ncbi.nlm.nih.gov ) . the potential kln coding sequences were tested using tests of reliability of eutherian public genomic sequences . the first test steps analysed nucleotide sequence coverages of each potential kln coding sequence , using ncbi 's blast programs and primary experimental genomic sequence reads deposited in ncbi 's trace archive ( https://www.ncbi.nlm.nih.gov/traces/trace.cgi ) . only if consensus read nucleotide sequence coverages were available for every nucleotide of each potential kln coding sequence , the potential kln coding sequences were annotated as complete kln coding sequences and used in analyses . alternatively , the potential kln coding sequences were described as putative kln coding sequences and not used in analyses . in addition , the complete kln coding sequences were deposited in european nucleotide archive as eutherian third party data gene data set ( http://www.ebi.ac.uk/ena/about/tpa-policy ) . the guidelines of human ( http://www.genenames.org/about/guidelines ) and mouse ( http://www.informatics.jax.org/mgihome/nomen/gene.shtml ) gene nomenclatures were used in updated kln gene nomenclature and classification . the mvista 's avid program was used in multiple pairwise genomic sequence alignments of kln genes using default settings ( http://genome.lbl.gov/vista/index.shtml ) . in major cluster klna alignments , the cut - offs of detection of common genomic sequence regions in alignments with base sequence ( homo sapiens klna1 ) were 70% per 100  bp except 65% per 100  bp in pairwise alignments between base sequence and rodents . the common predicted klna promoter genomic sequence regions were aligned at nucleotide sequence level using clustalw included in bioedit 7.0.5.3 , and manually corrected . using bioedit 7.0.5.3 , the pairwise nucleotide sequence identities of common predicted klna promoter genomic sequence regions were calculated and used in statistical analyses , including calculations of average pairwise identities (  ) and their average absolute deviations ( ad ) , largest pairwise identities ( amax ) and smallest pairwise identities ( amin ) ( microsoft office excel ) . in other major cluster alignments ( klnb - klnm ) , the cut - offs of detection of common genomic sequence regions in alignments with base sequences ( homo sapiens ) were : 95% per 100  bp ( pan troglodytes ) , 90% per 100  bp ( pongo abelii , nomascus leucogenys ) , 85% per 100  bp ( macaca mulatta , papio hamadryas ) , 80% per 100  bp ( callithrix jacchus ) , 75% per 100  bp ( otolemur garnettii ) , 65% per 100  bp ( rodents ) or 70% per 100  bp in other pairwise alignments , . the repeatmasker program version open-4.0.5 was used in detections and maskings of transposable elements in base sequences using default settings , except simple repeats and low complexity elements were not masked ( sensitive mode , cross_match version 1.080812 , repbase update 20140131 and rm database version 20140131 ) ( http://www.repeatmasker.org/ ) . therefore , among 335 potential coding sequences , the tests of reliability of eutherian public genomic sequences annotated most comprehensive curated third party data gene data set of eutherian kln genes including 121 complete coding sequences ( fig . 1a ) ( supplementary data file 1 ) . under accession numbers lt631550lt631670 , the eutherian kln gene data set was deposited in european nucleotide archive ( https://www.ebi.ac.uk/ena/data/view/lt631550-lt631670 ) . first , the major cluster klna included 26 genes ( klk1klk3 ) ( supplementary data file 2a ) . the common predicted klna promoter genomic sequence region was annotated , having average pairwise nucleotide sequence identities   =  0.74 ( amax  =  0.971 , amin  =  0.597 , ad  =  0.07 ) ( supplementary data file 3 ) . whereas the major cluster klnb included 10 genes ( klk4 ) ( supplementary data file 2b ) , major cluster klnc included 4 genes ( klk5 ) ( supplementary data file 2c ) and major gene cluster klnd included 7 genes ( klk7 ) ( supplementary data file 2d ) . the major cluster klne included 4 genes ( klk15 ) ( supplementary data file 2e ) , major gene cluster klnf included 11 genes ( klk9 ) ( supplementary data file 2f ) , major cluster klng included 10 genes ( klk11 ) ( supplementary data file 2 g ) and major cluster klnh included 12 genes ( klk8 ) ( supplementary data file 2h ) . whereas the major cluster klni included 5 genes ( klk6 ) ( supplementary data file 2i ) , major cluster klnj included 8 genes ( klk10 ) ( supplementary data file 2j ) and major gene cluster klnk included 11 genes ( klk12 ) ( supplementary data file 2k ) . finally , the major cluster klnl included 6 genes ( klk13 ) ( supplementary data file 2l ) and major cluster klnm included 7 genes ( klk14 ) ( supplementary data file 2 m ) . the present gene annotations confirmed major criteria of eutherian kln gene annotations including genomic localization within single genomic kallikrein locus region and common kln gene features including 5 translated exons , as well as different patterns of kln differential gene expansions between eutherians , , , , , , , , . of note , whereas the human kln gene annotations including 15 genes within kallikrein locus were likely complete , other kallikrein locus gene number estimates were subject to future updates and refinements due to incomplete genomic sequence assemblies and potential genomic sequence errors ( supplementary data file 1 ) . next , using bioedit 7.0.5.3 , the complete kln coding sequences were translated and aligned at amino acid level using clustalw included in bioedit 7.0.5.3 . after manual corrections of protein amino acid sequence alignments , phylogenetic tree calculations , the mega 6.06 program was used ( http://www.megasoftware.net ) , using neighbour - joining method ( default settings , except gaps / missing data treatment  =  pairwise deletion ) , minimum evolution method ( default settings , except gaps / missing data treatment  =  pairwise deletion ) , maximum parsimony method ( default settings , except gaps / missing data treatment  =  use all sites ) and unweighted pair group method with arithmetic mean method ( default settings , except gaps / missing data  =  pairwise deletion ) . however , the maximum likelihood methods were not used in present analysis because their homogeneity and stationarity assumptions were not satisfied ( data not shown ) . using bioedit 7.0.5.3 , the pairwise nucleotide sequence identities of kln nucleotide sequence alignments were calculated and used in statistical analyses , including calculations of average pairwise identities (  ) and their average absolute deviations ( ad ) , largest pairwise identities ( amax ) and smallest pairwise identities ( amin ) ( microsoft office excel ) . therefore , the present phylogenetic analysis described 13 major gene clusters of eutherian kln genes , using phylogenetic tree calculations ( fig . 1a ) and calculations of pairwise nucleotide sequence identity patterns ( supplementary data file 4 ) . for example , the 15 human kln genes were distributed among 13 major gene clusters of eutherian kln genes . the major eutherian kln protein sequence alignment landmarks included protein primary structure features ( fig . , whereas the major gene clusters klna and klnb included evidence of differential gene expansions respectively , major gene clusters klnc - klnm included orthologues respectively . there were major disagreements between present phylogenetic classification of eutherian kln genes and analyses of yousef et al . , pavlopoulou et al . , marques et al . , clements et al .   and koumandou and scorilas . specifically , the major gene cluster klna first integrated differentially expanded human klk1klk3 genes with differentially expanded rodent kln genes . however , the clustering of major gene cluster klnb ( klk4 ) with major gene cluster klnc ( klk5 ) agreed with analyses of yousef et al . yet , the clustering of major gene clusters klne ( klk15 ) , klnf ( klk9 ) and klng ( klk11 ) agreed with analyses of yousef et al . , elliott et al . , fernando et al . , but disagreed with analyses of pavlopoulou et al .   and koumandou and scorilas . finally , the clustering of major gene cluster klnl ( klk13 ) with major gene cluster klnm ( klk14 ) disagreed with analyses of yousef et al . , elliott et al . , fernando et al . , pavlopoulou et al .   and koumandou and scorilas . the present phylogenetic classification of eutherian kln genes was confirmed by calculations of pairwise nucleotide sequence identity patterns among 13 major gene clusters ( supplementary data file 4 ) . first , the complete kln nucleotide sequence alignments of 121 close eutherian homologues included average pairwise nucleotide sequence identity   =  0.473 ( amax  =  0.994 , amin  =  0.266 , ad  =  0.066 ) . among 13 eutherian kln major gene clusters respectively , there were nucleotide sequence identity patterns of close eutherian orthologues and paralogues ( klna and klnb ) and typical eutherian orthologues ( klnc - klnm ) . in comparisons between eutherian kln major gene clusters , there were nucleotide sequence identity patterns of very close eutherian homologues in comparisons between major gene clusters klnf , klng and klnh , in comparisons between major gene clusters klne and klng , as well as in comparisons between major gene clusters klni and klnm . finally , there were nucleotide sequence identity patterns of close eutherian homologues in other comparisons between major gene clusters . the tests of protein molecular evolution included complete kln nucleotide sequence alignments of 121 close eutherian homologues ( supplementary data file 5 ) . the mega 6.06 program was used in calculations of kln codon usage statistics . the relative synonymous codon usage statistics ( r ) there were 22 not preferable amino acid codons ( r    0.7 ) , including ttt ( 0,47 ) , tta ( 0,1 ) , ttg ( 0,46 ) , ctt ( 0,55 ) , cta ( 0,32 ) , att ( 0,61 ) , ata ( 0,37 ) , gtt ( 0,53 ) , gta ( 0,22 ) , tcg ( 0,37 ) , ccg ( 0,46 ) , act ( 0,61 ) , acg ( 0,48 ) , gcg ( 0,43 ) , tat ( 0,47 ) , cat ( 0,5 ) , caa ( 0,48 ) , aat ( 0,61 ) , gat ( 0,65 ) , gaa ( 0,56 ) , cgt ( 0,49 ) and agt ( 0,45 ) . using protein and nucleotide sequence alignments , the reference human klna2 ( prostate - specific antigen ) protein sequence amino acid sites were then described as invariant amino acid sites ( invariant alignment positions ) , forward amino acid sites ( variant alignment positions not including amino acid codons with r    0.7 ) or compensatory amino acid sites ( variant alignment positions including amino acid codons with r    0.7 ) . in kln protein primary structures , the signalp 4.1 server was used in predictions of n - terminal signal peptides using default settings ( http://www.cbs.dtu.dk/services/signalp/ ) . therefore , the eutherian kln proteins were described as extracellular peptidases , including pro - enzyme or zymogen protein activation after cleavage of n - terminal signal peptides , ,   ( supplementary data file 6 ) . the major protein sequence alignment landmarks comprised mature eutherian kln protein primary structure features , including common cysteine amino acid residues and n - glycosylation sites , as well as common exon - intron splice site amino acid sites ( fig . for example , there were 1013 cysteine amino acid residues common to eutherian kln major protein clusters respectively , including 10 invariant cysteine amino acid residues ,   ( supplementary data file 6 ) . in addition , the eutherian kln major protein clusters included 04 common n - glycosylation sites respectively , , . finally , the tests of protein molecular evolution integrated patterns of nucleotide sequence similarities with protein primary structures using present protein and nucleotide sequence alignments , ,   ( supplementary data file 5 ) specifically , among 261 reference human klna2 amino acid residues , the present analysis described 27 invariant amino acid sites including 10 invariant cysteine amino acid residues , as well as 6 forward amino acid sites ( supplementary data file 6 ) . for example , the invariant amino acid sites included catalytic triad amino acid sites his , asp and ser ( h65 , d120 and s213 in reference human klna2 protein primary structure ) , , , , . finally , there was one amino acid site cluster comprising overrepresented invariant and forward amino acid sites ( c209c219 in reference human klna2 protein primary structure ) . for example , the amino acid site cluster included 2 invariant cysteine amino acid residues , catalytic triad amino acid site ser , as well as amino acid motif gdsggp common to serine peptidases ,   and . the eutherian kallikrein genes were implicated in major physiological and pathological processes , as well as in medical molecular diagnostics , , , . for example , the serum level of human prostate - specific antigen abundant in seminal plasma was used as molecular marker in diagnostics of prostate cancer . the comprehensive eutherian kallikrein gene data sets established major criteria of kallikrein gene annotations , including genomic localization within single genomic kallikrein locus region , common kallikrein gene features including 5 translated exons , as well as patterns of kallikrein protein amino acid sequence similarities including invariant catalytic triad amino acid sites his , asp and ser and distribution of common cysteine amino acid residues , , , , , , , , , , . specifically , the human kallikreins comprised 15 serine peptidases , including 3 classical kallikreins klk1 or paradigmatic pancreatic kallikrein , klk2 or glandular kallikrein and klk3 or prostate - specific antigen , as well as klk4klk15 kallikrein - related serine peptidases . nevertheless , future updates and revisions of comprehensive eutherian gene data sets were expected , due to the incompleteness of public eutherian genomic sequence data sets ,   and potential genomic sequence errors . for example , the so - called lexicographical bias was described in some genomic sequence assembly programs   and phylogenetic analyses could be affected by potential genomic sequence errors . thus , the eutherian comparative genomic analysis protocol applicable in curation of major eutherian gene data sets was established as assistance in protection against potential genomic sequence errors , , . the protocol integrated gene annotations , phylogenetic analysis and protein molecular evolution analysis with new genomics and protein molecular evolution tests into one framework of eutherian gene descriptions . for example , the protocol revised , updated and published 9 major eutherian gene data sets , including 1172 complete coding sequences deposited in european nucleotide archive as curated third party data gene data sets . using eutherian comparative genomic analysis protocol and public genomic sequence assemblies , , , the present study made attempts to update and revise eutherian kallikrein kln genes . the sequence alignment editor bioedit 7.0.5.3 was used in analyses and manipulations of kln nucleotide and protein sequences ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) . the public eutherian genomic sequence assemblies used in gene identifications of potential kln coding sequences were downloaded from national center for biotechnology information ( ncbi ) genbank ( ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/vertebrate_mammalian/ ) , as well as ncbi 's blast programs ( ftp://ftp.ncbi.nlm.nih.gov/blast/ ) . in addition , the public eutherian genomic sequences were downloaded from ensembl genome browser ( http://www.ensembl.org ) . the analyses of kln gene features included direct evidence of gene annotations deposited in ncbi 's nr , est_human , est_mouse and est_others databases ( https://www.ncbi.nlm.nih.gov ) . the potential kln coding sequences were tested using tests of reliability of eutherian public genomic sequences . the first test steps analysed nucleotide sequence coverages of each potential kln coding sequence , using ncbi 's blast programs and primary experimental genomic sequence reads deposited in ncbi 's trace archive ( https://www.ncbi.nlm.nih.gov/traces/trace.cgi ) . only if consensus read nucleotide sequence coverages were available for every nucleotide of each potential kln coding sequence , the potential kln coding sequences were annotated as complete kln coding sequences and used in analyses . alternatively , the potential kln coding sequences were described as putative kln coding sequences and not used in analyses . in addition , the complete kln coding sequences were deposited in european nucleotide archive as eutherian third party data gene data set ( http://www.ebi.ac.uk/ena/about/tpa-policy ) . the guidelines of human ( http://www.genenames.org/about/guidelines ) and mouse ( http://www.informatics.jax.org/mgihome/nomen/gene.shtml ) gene nomenclatures were used in updated kln gene nomenclature and classification . the mvista 's avid program was used in multiple pairwise genomic sequence alignments of kln genes using default settings ( http://genome.lbl.gov/vista/index.shtml ) . in major cluster klna alignments , the cut - offs of detection of common genomic sequence regions in alignments with base sequence ( homo sapiens klna1 ) were 70% per 100  bp except 65% per 100  bp in pairwise alignments between base sequence and rodents . the common predicted klna promoter genomic sequence regions were aligned at nucleotide sequence level using clustalw included in bioedit 7.0.5.3 , and manually corrected . using bioedit 7.0.5.3 , the pairwise nucleotide sequence identities of common predicted klna promoter genomic sequence regions were calculated and used in statistical analyses , including calculations of average pairwise identities (  ) and their average absolute deviations ( ad ) , largest pairwise identities ( amax ) and smallest pairwise identities ( amin ) ( microsoft office excel ) . in other major cluster alignments ( klnb - klnm ) , the cut - offs of detection of common genomic sequence regions in alignments with base sequences ( homo sapiens ) were : 95% per 100  bp ( pan troglodytes ) , 90% per 100  bp ( pongo abelii , nomascus leucogenys ) , 85% per 100  bp ( macaca mulatta , papio hamadryas ) , 80% per 100  bp ( callithrix jacchus ) , 75% per 100  bp ( otolemur garnettii ) , 65% per 100  bp ( rodents ) or 70% per 100  bp in other pairwise alignments , . the repeatmasker program version open-4.0.5 was used in detections and maskings of transposable elements in base sequences using default settings , except simple repeats and low complexity elements were not masked ( sensitive mode , cross_match version 1.080812 , repbase update 20140131 and rm database version 20140131 ) ( http://www.repeatmasker.org/ ) . therefore , among 335 potential coding sequences , the tests of reliability of eutherian public genomic sequences annotated most comprehensive curated third party data gene data set of eutherian kln genes including 121 complete coding sequences ( fig . lt631550lt631670 , the eutherian kln gene data set was deposited in european nucleotide archive ( https://www.ebi.ac.uk/ena/data/view/lt631550-lt631670 ) . first , the major cluster klna included 26 genes ( klk1klk3 ) ( supplementary data file 2a ) . the common predicted klna promoter genomic sequence region was annotated , having average pairwise nucleotide sequence identities   =  0.74 ( amax  =  0.971 , amin  =  0.597 , ad  =  0.07 ) ( supplementary data file 3 ) . whereas the major cluster klnb included 10 genes ( klk4 ) ( supplementary data file 2b ) , major cluster klnc included 4 genes ( klk5 ) ( supplementary data file 2c ) and major gene cluster klnd included 7 genes ( klk7 ) ( supplementary data file 2d ) . the major cluster klne included 4 genes ( klk15 ) ( supplementary data file 2e ) , major gene cluster klnf included 11 genes ( klk9 ) ( supplementary data file 2f ) , major cluster klng included 10 genes ( klk11 ) ( supplementary data file 2 g ) and major cluster klnh included 12 genes ( klk8 ) ( supplementary data file 2h ) . whereas the major cluster klni included 5 genes ( klk6 ) ( supplementary data file 2i ) , major cluster klnj included 8 genes ( klk10 ) ( supplementary data file 2j ) and major gene cluster klnk included 11 genes ( klk12 ) ( supplementary data file 2k ) . finally , the major cluster klnl included 6 genes ( klk13 ) ( supplementary data file 2l ) and major cluster klnm included 7 genes ( klk14 ) ( supplementary data file 2 m ) . the present gene annotations confirmed major criteria of eutherian kln gene annotations including genomic localization within single genomic kallikrein locus region and common kln gene features including 5 translated exons , as well as different patterns of kln differential gene expansions between eutherians , , , , , , , , . of note , whereas the human kln gene annotations including 15 genes within kallikrein locus were likely complete , other kallikrein locus gene number estimates were subject to future updates and refinements due to incomplete genomic sequence assemblies and potential genomic sequence errors ( supplementary data file 1 ) . next , using bioedit 7.0.5.3 , the complete kln coding sequences were translated and aligned at amino acid level using clustalw included in bioedit 7.0.5.3 . after manual corrections of protein amino acid sequence alignments , phylogenetic tree calculations , the mega 6.06 program was used ( http://www.megasoftware.net ) , using neighbour - joining method ( default settings , except gaps / missing data treatment  =  pairwise deletion ) , minimum evolution method ( default settings , except gaps / missing data treatment  =  pairwise deletion ) , maximum parsimony method ( default settings , except gaps / missing data treatment  =  use all sites ) and unweighted pair group method with arithmetic mean method ( default settings , except gaps / missing data  =  pairwise deletion ) . however , the maximum likelihood methods were not used in present analysis because their homogeneity and stationarity assumptions were not satisfied ( data not shown ) . using bioedit 7.0.5.3 , the pairwise nucleotide sequence identities of kln nucleotide sequence alignments were calculated and used in statistical analyses , including calculations of average pairwise identities (  ) and their average absolute deviations ( ad ) , largest pairwise identities ( amax ) and smallest pairwise identities ( amin ) ( microsoft office excel ) . therefore , the present phylogenetic analysis described 13 major gene clusters of eutherian kln genes , using phylogenetic tree calculations ( fig . 1a ) and calculations of pairwise nucleotide sequence identity patterns ( supplementary data file 4 ) . for example , the 15 human kln genes were distributed among 13 major gene clusters of eutherian kln genes . the major eutherian kln protein sequence alignment landmarks included protein primary structure features ( fig . , whereas the major gene clusters klna and klnb included evidence of differential gene expansions respectively , major gene clusters klnc - klnm included orthologues respectively . there were major disagreements between present phylogenetic classification of eutherian kln genes and analyses of yousef et al . , elliott et al . , fernando et al . , lundwall and brattsand , lawrence et al . , pavlopoulou et al . , marques et al . , clements et al .   and koumandou and scorilas . specifically , the major gene cluster klna first integrated differentially expanded human klk1klk3 genes with differentially expanded rodent kln genes . however , the clustering of major gene cluster klnb ( klk4 ) with major gene cluster klnc ( klk5 ) agreed with analyses of yousef et al . , elliott et al . , fernando et al . , pavlopoulou et al .   and koumandou and scorilas yet , the clustering of major gene clusters klne ( klk15 ) , klnf ( klk9 ) and klng ( klk11 ) agreed with analyses of yousef et al . , elliott et al . , fernando et al . , but disagreed with analyses of pavlopoulou et al .   and koumandou and scorilas . finally , the clustering of major gene cluster klnl ( klk13 ) with major gene cluster klnm ( klk14 ) disagreed with analyses of yousef et al . , elliott et al . , fernando et al . , pavlopoulou et al .   and koumandou and scorilas . the present phylogenetic classification of eutherian kln genes was confirmed by calculations of pairwise nucleotide sequence identity patterns among 13 major gene clusters ( supplementary data file 4 ) . first , the complete kln nucleotide sequence alignments of 121 close eutherian homologues included average pairwise nucleotide sequence identity   =  0.473 ( amax  =  0.994 , amin  =  0.266 , ad  =  0.066 ) . among 13 eutherian kln major gene clusters respectively , there were nucleotide sequence identity patterns of close eutherian orthologues and paralogues ( klna and klnb ) and typical eutherian orthologues ( klnc - klnm ) . in comparisons between eutherian kln major gene clusters , there were nucleotide sequence identity patterns of very close eutherian homologues in comparisons between major gene clusters klnf , klng and klnh , in comparisons between major gene clusters klne and klng , as well as in comparisons between major gene clusters klni and klnm . finally , there were nucleotide sequence identity patterns of close eutherian homologues in other comparisons between major gene clusters . the tests of protein molecular evolution included complete kln nucleotide sequence alignments of 121 close eutherian homologues ( supplementary data file 5 ) . the mega 6.06 program was used in calculations of kln codon usage statistics . the relative synonymous codon usage statistics ( r ) there were 22 not preferable amino acid codons ( r    0.7 ) , including ttt ( 0,47 ) , tta ( 0,1 ) , ttg ( 0,46 ) , ctt ( 0,55 ) , cta ( 0,32 ) , att ( 0,61 ) , ata ( 0,37 ) , gtt ( 0,53 ) , gta ( 0,22 ) , tcg ( 0,37 ) , ccg ( 0,46 ) , act ( 0,61 ) , acg ( 0,48 ) , gcg ( 0,43 ) , tat ( 0,47 ) , cat ( 0,5 ) , caa ( 0,48 ) , aat ( 0,61 ) , gat ( 0,65 ) , gaa ( 0,56 ) , cgt ( 0,49 ) and agt ( 0,45 ) . using protein and nucleotide sequence alignments , the reference human klna2 ( prostate - specific antigen ) protein sequence amino acid sites were then described as invariant amino acid sites ( invariant alignment positions ) , forward amino acid sites ( variant alignment positions not including amino acid codons with r    0.7 ) or compensatory amino acid sites ( variant alignment positions including amino acid codons with r    0.7 ) . in kln protein primary structures , the signalp 4.1 server was used in predictions of n - terminal signal peptides using default settings ( http://www.cbs.dtu.dk/services/signalp/ ) . therefore , the eutherian kln proteins were described as extracellular peptidases , including pro - enzyme or zymogen protein activation after cleavage of n - terminal signal peptides , ,   ( supplementary data file 6 ) . the major protein sequence alignment landmarks comprised mature eutherian kln protein primary structure features , including common cysteine amino acid residues and n - glycosylation sites , as well as common exon - intron splice site amino acid sites ( fig . for example , there were 1013 cysteine amino acid residues common to eutherian kln major protein clusters respectively , including 10 invariant cysteine amino acid residues ,   ( supplementary data file 6 ) . in addition , the eutherian kln major protein clusters included 04 common n - glycosylation sites respectively , , . finally , the tests of protein molecular evolution integrated patterns of nucleotide sequence similarities with protein primary structures using present protein and nucleotide sequence alignments , ,   ( supplementary data file 5 ) . specifically , among 261 reference human klna2 amino acid residues , the present analysis described 27 invariant amino acid sites including 10 invariant cysteine amino acid residues , as well as 6 forward amino acid sites ( supplementary data file 6 ) . for example , the invariant amino acid sites included catalytic triad amino acid sites his , asp and ser ( h65 , d120 and s213 in reference human klna2 protein primary structure ) , , , , . in contrast , the analysis of clements et al .   described 39 invariant amino acid sites in human kln protein primary structures . finally , there was one amino acid site cluster comprising overrepresented invariant and forward amino acid sites ( c209c219 in reference human klna2 protein primary structure ) . for example , the amino acid site cluster included 2 invariant cysteine amino acid residues , catalytic triad amino acid site ser , as well as amino acid motif gdsggp common to serine peptidases ,   and . in conclusion , the most comprehensive curated third party data gene data set of eutherian kln genes first annotated 13 major gene clusters klna - klnm and explained their differential gene expansion patterns . as new framework of future experiments , the gene annotations , phylogenetic analysis and protein molecular evolution analysis integrated with new genomics and protein molecular evolution tests proposed updated classification and nomenclature of eutherian kln genes . the following are the supplementary data related to this article.supplementary data file 1third party data gene data set of eutherian kallikrein genes.supplementary data file 1supplementary data file 2multiple pairwise genomic sequence alignments of eutherian kallikrein genes . the translated genomic sequence regions were displayed as indigo rectangles in base sequences ( top ) . the genomic sequence regions including sequence identity levels above empirical cut - offs of detection of common genomic sequence regions were shown accordingly in multiple pairwise alignments . ( a ) the common predicted promoter genomic sequence regions were labelled by rectangles ( p).supplementary data file 2supplementary data file 2supplementary data file 2supplementary data file 2supplementary data file 2supplementary data file 2supplementary data file 2supplementary data file 3nucleotide sequence alignments of common predicted promoter genomic sequence regions of eutherian klna genes . the positions of 3-terminal nucleotides relative to translation start sites were indicated by numbers in brackets . the nucleotide positions were labelled using white letters on black background ( 100% sequence identity level ) , white letters on dark grey background (   85% sequence identity level ) or black letters on grey background (   70% sequence identity level).supplementary data file 3supplementary data file 4pairwise nucleotide sequence identity patterns of eutherian kallikrein genes.supplementary data file 4supplementary data file 5protein sequence alignments of eutherian kallikreins . the amino acid positions were labelled using white letters on black background ( 100% sequence identity level ) , white letters on dark grey background (   75% sequence identity level ) or black letters on grey background (   50% sequence identity level ) . whereas the 27 invariant amino acid sites were shown using white letters on violet backgrounds , 6 forward amino acid site was shown using white letter on red background in reference human klna2 protein amino acid sequence ( top ) . the stop codons were indicated by & s.supplementary data file 5supplementary data file 6reference human klna2 protein primary structure . whereas the 27 invariant amino acid sites were shown using white letters on violet backgrounds , 6 forward amino acid sites were shown using white letters on red backgrounds . the 10 invariant cys amino acid residues were labelled above reference protein amino acid sequence ( 24 , 814 ) , as well as common exon - intron splice site amino acid residues ( # ) . in addition , the catalytic triad amino acid sites h65 , d120 and s213 were indicated by arrows above reference protein amino acid sequence , . the amino acid residues implicated in h bonding between human klna2 , fluorogenic substrate mu - kgissqy - afc and mab 8g8f5 were labelled below reference protein amino acid sequence ( x ) , . the amino acid site cluster c209c219 including overrepresented invariant and forward amino acid sites was indicated by rectangle . the grey triangle showed pro - enzyme protein activation cleavage site , , .supplementary data file 6 third party data gene data set of eutherian kallikrein genes . the translated genomic sequence regions were displayed as indigo rectangles in base sequences ( top ) . the genomic sequence regions including sequence identity levels above empirical cut - offs of detection of common genomic sequence regions were shown accordingly in multiple pairwise alignments . ( a ) the common predicted promoter genomic sequence regions were labelled by rectangles ( p ) . the positions of 3-terminal nucleotides relative to translation start sites were indicated by numbers in brackets . the nucleotide positions were labelled using white letters on black background ( 100% sequence identity level ) , white letters on dark grey background (   85% sequence identity level ) or black letters on grey background (   70% sequence identity level ) . the amino acid positions were labelled using white letters on black background ( 100% sequence identity level ) , white letters on dark grey background (   75% sequence identity level ) or black letters on grey background (   50% sequence identity level ) . whereas the 27 invariant amino acid sites were shown using white letters on violet backgrounds , 6 forward amino acid site was shown using white letter on red background in reference human klna2 protein amino acid sequence ( top ) . whereas the 27 invariant amino acid sites were shown using white letters on violet backgrounds , 6 forward amino acid sites were shown using white letters on red backgrounds . the 10 invariant cys amino acid residues were labelled above reference protein amino acid sequence ( 24 , 814 ) , as well as common exon - intron splice site amino acid residues ( # ) . in addition , the catalytic triad amino acid sites h65 , d120 and s213 were indicated by arrows above reference protein amino acid sequence , . the amino acid residues implicated in h bonding between human klna2 , fluorogenic substrate mu - kgissqy - afc and mab 8g8f5 were labelled below reference protein amino acid sequence ( x ) , . the amino acid site cluster c209c219 including overrepresented invariant and forward amino acid sites was indicated by rectangle .
the present study made attempts to update and revise eutherian kallikrein genes implicated in major physiological and pathological processes and in medical molecular diagnostics . using eutherian comparative genomic analysis protocol and free available genomic sequence assemblies , the tests of reliability of eutherian public genomic sequences annotated most comprehensive curated third party data gene data set of eutherian kallikrein genes including 121 complete coding sequences among 335 potential coding sequences . 
 the present analysis first described 13 major gene clusters of eutherian kallikrein genes , and explained their differential gene expansion patterns . 
 one updated classification and nomenclature of eutherian kallikrein genes was proposed , as new framework of future experiments .