Dating Doppel (PRND)

Dating doppel: missing from bird and fish genomes -- but present in marsupial, lizard and frog

The prion and doppel genes represent a local tandem duplication that has descended intact in most lineages with the two genes remaining adjacent and unpseudogenized (though both have spawned additional retroposed pseudogenes). When did this gene duplication take place, did it correlate with or facilitate an evolutionary advancement, and how have the (largely unknown) normal functions of the two genes since diverged? Retention of the second copy is somewhat puzzling in view of viability of PRNP knockouts, indeed ">double knockouts.

The weak homology between prion and doppel, below 30% identity even for the conserved core, suggests either a quite ancient duplication (lamprey era?) followed by average divergence, or alternatively, a fairly recent duplication (tetrapod era?) followed by rapid evolution on the doppel side as it acquired a auxillary function (neofunctionalized). These possibilities can be distinguished by looking at earlier diverging species that put a date on the duplication event.

If the duplication is old, then doppel should be found in various draft vertebrate genomes now available -- 34 mammals (see Genome completion status), chicken, lizard, frog, zebrafish, tetraodon, fugu, medaka, and stickleback. If gene order and orientation (synteny) are conserved, doppel will lie between PRNP and RASSF2, allowing highly diverged forms to be recognized that might give unconvincing blast matches. Of course synteny can be supplemented by conservation of internal signatures such as signal peptide, post-translationally modified arginine, disulfides, glycosylation sites, alpha helices, short beta sheet (YMLG in human), and GPI attachment. Repeat structure is trickier to use because it has been lost in all doppels and shifts from biphasic hexapeptide in frog, turtles, birds, and lizards to decapeptide (platypus), nonapeptide (marsupial), to octapeptide (placentals).

The brushtail opossum (Trichosurus vulpecular) prion gene sequence was determined in 1995. It conserves structural elements overall but presents various anomalies such as nonapeptide repeats. Its sequence can be used as query at the UCSC genome browser to find a complete second marsupial PRNP sequence from Monodelphis domestica. The two proteins have 86% sequence identity (not including repeat region), not necessarily indicating faster divergence since marsupials have had longer to diverge than placental mammals. The two 'opossums' here are not closely related, Didelphimorphia vs Diprotodontia. The second marsupial genome (Macropus eugenii) has been released unassembled to NCBI trace archives; full length PRNP and PRND sequences are readily covered there by trace blastn.

Monodelphis domestica PRND is located next to PRNP in the same orientation in a gapless region of the assembly. Within placental mammals, doppel homology is about 85% identity but this drops off to 46% relative to marsupial, indicating a relative fast-evolving protein in this lineage (but not yet a pseudogene). Indeed, with nearly PRNP gene sequences available in nearly a hundred species of placentals and the mammalian phylogenetic tree topology finally determined, clade-specific rate variations could be pursued in unusual detail, with a possible correlation with rates of doppel evolution.

Monotreme represents a still earlier divergence: PRNP and PRND can be recovered from the newly released assembly of the platypus genome, Ornithorhynchus anatinus. This species, which diverged after birds but before the Metatherian/Eutherian split, has a decapeptide x 4 repeat (rather than a bird- or turtle-like hexapeptide. However its sequence, PQGGGASWGH, is unusual for its glutamine in second position and terminal histidine (reverse of usual mammal residues). In other respects it is conventional.

The chicken genome is gap-free in this area and retains mammalian gene order and orientation (PRNP+, RASSF2-, and SCL23A-). However, no dopple gene occurs in chicken in the expected position. If prion gene duplication occurred prior to chicken divergence, then doppel decayed to an unrecognizable pseudogene, translocated to an unsequenced region, or has been deleted. No evidence for a decayed pseudogene is observed using a high-sensitivity blast of reconstructed ancestral doppel. The other bird genome underway, Taeniopygia guttata (finch), also lacks PRND.

Very recently, a lizard genome became available, Anolis carolinensis. Using specialized methods, a full length PRND gene has be located in expected syntenic position and validated by retained internal signatures despite its very weak blast score. Similarly in the diploid frog, Xenopus tropicalis, an unmistakable frog doppel gene occurs at expected position and orientation. These results have a simple parsimonious explanation: the PRND duplication predated amphibian divergence, followed by a lineage-specific deletion in the bird clade. Crocodile sequencing could refine the date of this event.

In support, lizard and frog doppel queries retrieve solely mammalian and turtle PRND genes when back-blasted against the GenBank non-redundant nucleotide division. This argues against the idea of multiple independent duplications of the prion gene in lizard and frog distinct from a later lineage-specific duplication in mammals (with birds then never having a PRND to delete).

Note the more familiar tetraploid laboratory frog, Xenopus laevis, has two actively transcribed PRNP genes that differ slightly in the repeat-like region as well at a few individual amino acids. Their sequences are given below. No sign of doppel can be found in Xenopus laevis cDNA (unsurprisingly since doppel transcription is rare); no genome project is underway. There may be two doppels in this species, depending on the relative timing of PRNP duplication and the tetraploidization event and selective pressure to maintain two copies.

The prion gene in teleost fish has radically diverged from its mammalian counterpart especially in the repeat region but remains recognizable via various conserved anchor features, residual synteny, and weak homology. Fugu has a second PRNP gene on a different chromosome (resulting from the whole genome duplication in Teleostei) plus an apparent novel tandem duplication that resembles PRNP rather than PRND. Coelocanth genome has been proposed but not begun -- that could further refine the timing of PRND duplication. As it stands, that event occurred after teleost fish divergence but before that of amphibians, about 400 million years ago. The prion gene clearly represents the ancestral form and presumably function, ie, a doppel-like ancestor did not duplicate to a second gene that become PRNP. No counterpart of PRNP can be located in the initial release of the chondrichthyian genome, Callorhynchus milii, but that will be finished to higher coverage in a few months.

Doppels are evolving noticeably faster than prions but have not become pseudogenes in any species investigated to date. (Note very recent pseudogenes that have not yet acquired internal stop codons or loss of deeply invariant residues are difficult to detect.) It must be remembered that PRNP's globular region lies within the most slowly evolving quartile of mammalian genes. As a representative example, human/dog proteins have 78% identity for PRND but 87% for PRNP. A reconstructed ancestral mammal sequence for PRND shows no sign whatsoever of converging to a PRNP-like sequence, percent identity ancester-to-ancestor remains in the mid-20's. Since this uses up 100 million years of the 400 available, doppel evolved more drastically at an earlier time, perhaps soon after the duplication event.

To further investigate the co-evolution of PRNP and PRND, it is imperative to first collect gene sequences from a phylogenetically representative sample of species. PRNP tends to be over-sampled within Artiodactylia, indeed Bovidae. Doppel remains very poorly represented at dbEST even within much-studied mammals and enriched libraries, even in testis, a tissue where it is transcribed. The most economical explanation of the data is that doppel has a narrow function with limited and specialized expression in uncommon cell types, yet one important enough for gene retention.

However a respectable number of tetrapod doppel sequences have accumulated. In addition to genome projects, van Rheede and coworkers sequenced nine doppels from important early diverging clades of placental mammals. PRND from the chimpanzee genome has special interest for evaluating known human doppel polymorphisms to determine "wildtype" human. Macaque doppel can also the 2 amino acid deletion seen in human at position 122-123, showing this event predated old world monkey divergence. In fact that event can be precisely timed to the stem lying between tarsier and new world monkey divergence and shown non-homplasic using various genome projects underway:

PRND_hsa  ANQGEFQ--KPDNKLHQQVLWRLVQELCS   Homo  sapiens  (human)
PRND_ptr  ANQGEFQ--KPDNKLHQQVLWRLVRELCS   Pan  troglodytes  (chimp)
PRND_ggo  ANQGEFH--KPDNKLHQQVLWRLVRELCS   Gorilla  gorilla  (gorilla)
PRND_ppy  ANQGEFQ--KPDNKLHQQVLWRLVQELCS   Pongo  pygmaeus  (orang_sumatran)
PRND_nle  ANQGEFQ--KPDNKLHQRVLWRLVRELCS   Nomascus  leucogenys  (gibbon)
PRND_mml  ENQGEFQ--KPDNKLHQRVLWRLVQELCS   Macaca  mulatta  (rhesus)
PRND_pha  ANQGEFQ--KPDNKLHQRVLWRLVQELCS   Papio  hamadryas  (baboon)
PRND_cja  ANQAEFQ--KPDNKLHQRVLWRLVQELCS   Callithrix  jacchus  (marmoset_nwm)
PRND_tsy  ANAAERPREARDDPLHQRVLGRLVRELCS   Tarsius  syrichta  (tarsier)
PRND_mmr  ANQAEFAREKQD-KLHERILWRLTRELCS   Microcebus  murinus  (mouse_lemur)
PRND_tbe  ANQAEFSKEKQDNKLYQRVLWRLIKELCS   Tupaia  belangeri  (tree_shrew)
PRND_ocu  ANQGEFSREKQDNKLHQRVLWRLIKELCS   Oryctolagus  cuniculus  (rabbit)
PRND_opr  ANQGEFSREKQ-NKLHQRVLWRLIKELCS   Ochotona  princeps  (pika)
PRND_dor  ANQAEFAREMQD-KFYQRVLWRLTKELCA   Dipodomys  ordii  (kangaroo_rat)
PRND_str  ANQAEFSREKQDNKLHQRVLWRLIKELCS   Spermophilus  tridecemlineatus  (ground_squirrel)
PRND_rno  ANQAEFSREKQDSKLHQRVLWRLIKEICS   Rattus  norvegicus  (rat)
PRND_mmu  ANQAEFSREKQDSKLHQRVLWRLIKEICS   Mus  musculus  (mouse)
PRND_cpo  ANQAEFSREKQDNKLHQRILWRLIKELCS   Cavia  porcellus    (guinea_pig)
PRND_sar  ANQEELSREKHD-KLYQRVLWRLVRELCS   Sorex  araneus  (shrew)
PRND_eeu  ANQEELSREKHD-KLYQRVLWRLVRELCS   Erinaceus  europaeus  (hedgehog)
PRND_cfa  ANQEELSREKQDNKLHQRVLWRLIRELCS   Canis  familiaris  (dog)
PRND_fca  ANQEELSREKQDDKLYQRVLWRLNR-ECS   Felis  catus  (cat)
PRND_tte  ENQEELSREKQDDKLHQRILWRLIRELCS   Tapirus  terrestris  (tapir)
PRND_eca  ANQEELSQEKQDDKLYQRILWRLISELCS   Equus  caballus  (horse)
PRND_mlu  ANQEELSQERPDNRLHRRILWRLVRELCS   Myotis  lucifugus  (microbat)
PRND_csp  VNQEELSQEKQDKLLHQRILWQLIRELCS   Cynopterus  sphinx  (bat)
PRND_oar  ANQEELSREKQDNKLYQRVLWQLIRELCS   Ovis  aries  (sheep)
PRND_chi  ANQEELSREKQDNKLYQRVLWQLIRELCS   Capra  hircus  (goat)
PRND_bta  ANQEELSREKQDNKLYQRVLWQLIRELCS   Bos  taurus  (cow)
PRND_ttr  VNQEELSREKQDNSLYQRVLWQLIRELCS   Tursiops  truncatus  (dolphin)
PRND_ssc  ANQEE-SHEKPDNKLYQRVLWRLIRELCS   Sus  scrofa  (pig)
PRND_pcw  VNQEELSLEKPDNKLYQRVLWRLIRELCS   Physeter  catodon  (whale)
PRND_ban  ANQEEFSREKQDNKLHQRVLWRLIRELCS   Boreoeuthere  ancestralis  (boreoeuthere)
PRND_dno  ANQAELAHERQD-TLHGRVLGRLIRELCA   Dasypus  novemcinctus  (armadillo)
PRND_cho  ANQAEFSREKHD-KLHQRVLWRLIRELCS   Choloepus  hoffmanni  (sloth)
PRND_pcp  VNQEEFSREKQDNKLYQRILWRLIRELCS   Procavia  capensis  (hyrax)
PRND_ete  ANQAEFSAPQQDSRLHQRVLWRLIRELCS   Echinops  telfairi  (tenrec)
PRND_ema  ANQEEFSREKQDNKVYQRILWRLIRELCS   Elephas  maximus  (elephant)
PRND_laf  ANQEEFSR-KQDNKVYQRILWRLIRELCS   Loxodonta  africana  (elephant)
PRND_tma  ANQEEFSREKQDNKLYQRILWRLIRELCS   Trichechus  manatus  (manatee)
PRND_mdo  INKLEPLEEQNISDIYSRILEQLIKELCA   Monodelphis  domestica  (opossum)
PRND_meu  VNNLETLEEKNASDIHSRVLEQLIKELCA   Macropus  eugenii  (wallaby)
PRND_oan  ANRGELTAGGNASDVHVRVLLRLVEELCA   Ornithorhynchus  anatinus  (platypus)
PRND_aca  SNKLNLSKGKDLSNTKERVMWVLIHHLCA   Anolis  carolinensis  (lizard)
PRND_xtr RNKVWISQLEDDEEGDIYMSVATQVLQFLCM  Xenopus  tropicalis  (frog)
           ** * .  * *. ::*::**:* :* .:    

Below 45 PRND doppel sequences from land vertebrates are provided. Most are complete and reliable, but others are fragmentary and no better than the ongoing genome project from which they were extracted (initially low coverage, perhaps a single applicable trace). The phylogenetic distribution is approximately half as extensive as for PRNP. Almost all species having an available PRND would also have an available PRNP. It would be very easy to extend PRND coverage since primer design for PCR presents no issues. All sequences consist of a single coding intron. The only rare genomic event for PRND of any phylogenetic depth is the indel shown above.

11 primates (Euarchonta)
7 glires (rodents and rabbits)
14 laurasiatheres
1 ancestral boreoeuthere
2 xenarthrans
5 aftrotheres
2 marsupial
1 platypus
0 bird
1 lizard
1 amphibian

>PRND_hsa human full length
MRKHLSWWWLATVCMLLFSHLSAVQTRGIKHRIKWNRKALPSTAQITEAQVAENRPGAFIKQGRKLDIDFGAEGNRYY
EANYWQFPDGIHYNGCSEANVTKEAFVTGCINATQAANQGEFQKPDNKLHQQVLWRLVQELCSLKHCEFWLERGAGLRVTMHQPVLLCLLALIWLTVK*

>PRND_ptr Pan chimpanzee full length
MRKHLSWWWLATVCMLLFSHLSAVQARGIKHRIKWNRKALPSTAQITEAQVAENRPGAFIKQGRKLDIDFGAEGNRYY
EANYWQFPDGIHYNGCSEANVTKEVFVTGCIHATQAANQGEF
QKPDNKLHQQVLWRLVRELCSLKHCEFWLERGAGLRVTMHQPVLLCLLAFIWLMVK*

>PRND_ggo gorilla
MRKHLSWWWLATVCMLFFSHLSAVQARGIKHRIKWNRKALPSTAQITEAQVAENRPGAFIKQGRKLDIDFGAEGNRYY
EANYWQFPDGIHYNGCSEANVTKEVFVTGCINATQAANQGEF
HKPDNKLHQQVLWRLVRELCSLKHCEWLERSAGLRVTMHQPVLLCLLAFIWLMMK*

>PRND_ppy Pongo pygmaeus orangutan shares 2 bp deletion unique to primates.
MKNHLSWWLATVCMLLFSHLSAVQARGIKHRIKWNRKALPSTAQITEAPGAENRPGALSkQGR  
QLDIDFGTEGNRYYEANYWQFPDGIHYDGCSEANVTKEVFVTGCINATQAANQGEFQ
KPDNKLHQQVLWRLVQELCSLKHCEFWLERGAGLRVTMHQPVLLCLLAFIWLMVK*

>PRND_nle Nomascus leucogenys gibbon
MRKHLSWWWLATVCMLLFSHLSAVQARGIKHRIKWNRKALPSTAQITEAQVAENRPGGFIKQGRKLDIDFGAEGNRYY
EANYWQFPDGIHYDGCSEANVTKEVFV
TGCINATQAANQGEFQKPDNKLHQRVLWRLVRELCSLKRCEFWLERGAGLRVTMHQPVLLCLLAFIWLMVK*

>PRND_pha Papio hamadryas (baboon)
        WLSRLHAAPQ HLSLVVQARGIKHRIKWNRKALPSTAQITEAQVAENPPGSFIKQGRKLNIDFGAEGNRYY
EANYWQFPDGIHYDGCSEANVTKEVFVTGC
INATQAANQGEFQKPDNKLHQRVLWRLVQELCSLKRCEFWLEGGAGLRVTMHQPVLLCLPAFVWLMVK*

>PRND_mml Macaca mulata full length from trace archives
MRQHLSRWWLATVCMLLLSHLSVVQARGIKHRIKWNRKALPSTAQITEAQVAENRPGAFIKQGR
KLNIDFGAEGNRYYEANYWQFPDGIHYDGCSEANVTKEVFVTGCINATQAENQGEFQK
PDNKLHQRVLWRLVQELCSLKRCEFWLERGAGLRVTMHQPVLLCLPAFVWLMV*

>PRND_cja Callithrix jacchus 1st new world monkey 89%  
MRKHLSGWWLATVCMLLFSHLSAVQARGIKHRIKWNRKALPSAAQITEAQVAENRPGTFIKQGRKLDINFGAEGNRYY
EANYWQLPDGIHYDGCSETNVTKEVFVTGCINATQAANQAEFQKPDNKLHQRVLWRLVQELCSLKRCEFWLERGAGFRVATHQPLLLCLLAFIWLMAK*

>PRND_tsy Tarsius syrichta tarsier 
MRKPLGGWRLAVVCVLLLGLLSAARARGIKHRFKWNRKASPSAARVTEARVAESRPGAFIRRGRKLDIDFGAEGNKYY
EANYWQFPDGIHYDGCSKANVTKELLVAGCINATHAANAAERPREARDDPLHQRVLGRLVRELCSAKHCDFWPERAAAPGTARARPGALLLVSRL

>PRND_mmr Microcebus murinus
GNRYYEANYWQFPDGIHYDGCSDANVTKEVFVASCINATVAANQAEFAREKQDKLHERILWRLTRELCSVKRCEFWLERGAGLRVAEDCPTILSLLLLLWLLLR*

>PRND_tbe Tupaia belangeri tree shrew 6.7 m tr frag
RALPSTAHITEARVAENRPGAFIRQGRKLDIDFGVEGNRYYEANYWQFPDGIHYDGCSEVNVTKEMFITSCINATQA
ANQAEFSKEKQDNKLYQRVLWRLIKELCSIKHCDFWLERGAGLRVTMHLPVMLCLLVFVWFVVK*

>PRND_mmu Mus musculus mouse full length
MKNRLGTWWVAILCMLLASHLSTVKARGIKHRFKWNRKVLPSSGGQITEARVAENRPGAF
IKQGRKLDIDFGAEGNRYYAANYWQFPDGIYYEGCSEANVTKEMLVTSCVNATQAANQAE
FSREKQDSKLHQRVLWRLIKEICSAKHCDFWLERGAALRVAVDQPAMVCLLGFVWFIVK*
 
>PRND_rno Rattus norvegicus rat full length
MKNRVGTWGLAILCLLLASHLSTVKARGIKHRFKWNRKVLPSSGQITEAQV
AENRPGAFIKQGRKLDIDFGAEGNKYYAANYWQFPDGIYYEGCSEANVTKEVLVTRCVNA
TQAANQAEFSREKQDSKLHQRVLWRLIKEICSTKHCDFWLERGAALRITVDQQAMVCLLGFIWFIVK*

>PRND_cpo Cavia porcellus (guinea pig) fragment AY130773 N-extended at traces 7,499,469 seq
MKTSLCVWALVCVLLQCHFSSVAARGIKHRIRWGRKPTPSPSQVTEARVAMTRPGAFIKEGHKLNIDFGAEGNRYYETNYWQFPDGIHYNGCSDT
NVTKEVLVTSCINATQAANQAEFSREKQDNKLHQRILWRLIKELCSA

>PRND_dor Dipodomys ordii pseudogene
ITEARYRENAQGPSSIKP--QADIDF--ERAQ*YEA*YWQFADGINYEGCSRRHVTKQMF
VSRCINVTQAANQAEFAREMQD-KFYQRVLWRLTKELCAVKQCDFWLERGEGCPVAIVNR
PAMLGLLLCMWFIL

>PRND_str Spermophilus tridecemlineatus ground squirrel complete tr
MKKHLGTWGLAVVCVLLSTHLSAIKARGIKHRIKWNRKSVPSTVQITEAQVAQNPPGAF
IKQGRKLHIDFGAEGNKYYEANYWQFPDGIYYDGCSEANVTKEVFIAKCINATQAANQAE
FSREKQDNKLHQRVLWRLIKELCSVKHCDFWLEGGAGFQLSVDQPVMLCLLVFIWLMVK*

>PRND_ocu Oryctolagus cuniculus rabbit revised complete from traces 8,968,713 sequences 75%
MRKHLGAWGLAIVCVLLFSHLPPAKARGIKHRIKWNRKALPSTAQITEAHVAENRPGTFI
RQGRKLNIDFGPEGNKYYEANYWQFPDGVHYDGCSEGNVTKEAFVAKCVNATQAANQGE
FSREKQDNKLHQRVLWRLIKELCSIKHCDFWLERGAGVQVGGEQPLVLGLLLCTWLLVK*

>PRND_opr Ochotona princeps pika complete
MRKHLGGWGLAIVCVLLFSLLPTVKARGIKHRIKWNRKALPSTAQITEARVAENRPGTFIRQGQKLSIDFGPEGNKYY
EANYWQFPDGIHYDGCSEGNVTKEVFVAKCINATQAANQGEFSREKQNKLHQRVLWRLIKELCSIKHCDFWLERGAGFPVTVGQPMVVLCLLIFTWVLVK*

>PRND_cfa Canis familiaris dog full length
MRKHLGGCWLAIVCVLLLSQLSAVEARGIKHRIKWNRKALPGTSQVTEARSAEIRPGAFI
RQGRKLDIDLGPEGNRYYEANYWQFPDGIHYNGCSEANVTKEKFVTGCINATQVANQEEL
SREKQDNKLHQRVLWRLIRELCSVKRCDFWLERGAGPRVAGAQPVLLCLLAFIWFIVK*

>PRND_fca Felis catus cat improved fragment  8,248,053 traces now 9.2
MRKHLGGCWLAIVCVLLFSQLSAVKARGIKHRIKWNRKTLPSISQVTEAHTAEIRPGAFIRQGRKLDIDLG
AEGNRYYEANYWQFPDGIHYNGCSEANVTKEKFVTSCINATQVANQEELSREKQDhKLYQRVLWRLNREALLWSAHGNFGRERG

>PRND_eca Equus caballus perissodactyl
MRKHLGGCCLAIVCVLLFSQLPVVLSRGTKHRIKWNRKALPSTAKVTEARVEEIRPGAF
IRQGRKLNINFGAEGNRYYEANYWQFPDEIHYNGCSEANVTKEKFVISCINATQEANQEE
LSQEKQDDKLYQRILWRLISELCSVKHCDFGLESGTGLRVTMDQPVLLCLLVFIWFIVK*

>PRND_tte Tapirus terrestris (Brazilian tapir) perissodactyl fragment
KALPSTSKVTEAHTAEIRPGAFIRQGRKLNIDFGAEGNRYYEANYWQFPDEIHYNGCSEA
NVTKEKFVISCINATQAENQEELSREKQDDKLHQRILWRLIRELCSV 

>PRND_mlu Myotis lucifugus traces ti|983665423 complete
MRKQLGGCCLAIVCVLLFSQLAAVQARGIKHRIKWNRKGAAPPSSSQVTEAQRVEMQLRP
GAFIKHGRKLDIDFGAEANRYYEANYWQFPDEIHYNGCSSEANVTREKFVSGCINATQAA
NQEELSQERPDNRLHRRILWRLVRELCSVKHCDFWLENGAGLWVTVHRPVMPCLLVSIWFIVK*

>PRND_pva Pteropus vampyrus frameshifty frag
MRRQLGGCWLAIVCVLLFSQLSTVKARGIKHRTKWNR PCDRVS VTESQ GAFIKQGRK

>PRND_csp Cynopterus sphinx bat  Laurasiatheria; Chiroptera fragment
KTLPSPSSHVTESQTAEMRPGAFVKQGRKLNIDFGDEGNRYYEAHYWEFPDGIYYDACSKANVTKEK
FVTSCINATQAVNQEELSQEKQDKLLHQRILWQLIRELCSV

>PRND_bta Bos taurus cow full length
MRKHLGGCWLAIVCILLFSQLCSVKARGIKHRIKWNRKVLPSTSQVTEARTAEIRPGAFI
KQGRKLDIDFGVEGNRYYEANYWQFPDGIHYNGCSKANVTKEKFITSCINATQAANQEEL
SREKQDNKLYQRVLWQLIRELCSTKHCDFWLERGAGLRVTLDQPMMLCLLVFIWFIVK*

>PRND_oar Ovis aries sheep full length
MRKHLGGCWLAIVCVLLFSQLSSVKARGIKHRIKWNRKVLPSTSQVTEAHTAEIRPGAFI
KQGRKLDINFGVEGNRYYEANYWQFPDGIHYNGCSEANVTKEKFVTSCINATQVANQEEL
SREKQDNKLYQRVLWQLIRELCSIKHCDFWLERGAGLQVTLDQPMMLCLLVFIWFIVK*

>PRND_chi Capra hircus (goat)
MRKHLGGCWLAIVCVLLFSQLSSVKARGIKHRIKWNRKVLPSTSQVTEAHTAEIRPGAFIKQGRKLDINFGV
EGNRYYEANYWQFPDGIHYNGCSEANVTKEKFVTSCINATQVANQEELSREKQDNKLYQRVLWQLIREL
CSIKHCDFWLERGAGLQVTLDQPMMLCLLVFIWFIVK*

>PRND_ama Antidorcas marsupialis (springbok) Bovidae; Antilopinae
MRKHLGGCWLAIACILLFSQLSSVKARGIKHRIKWNRKVLPSTS
QVTEAHTAEIRPGAFIKQGRKLDIDFGVEGNRYYEANYWQFPDGIHYNGCSEANVTKE
KFVTSCINATQVANQEELSREKQDNKLYQRVLWQLIRELCSIKHCDFWLERGAGLRVT
LDQPMMLCLLVFIWFIVK*

>PRND_ttr Tursiops truncatus dolphin 1 trace ti|1435196145
MRKHLGGCWLAIVCVLLFSQLSSVKARGIKHRIKWNRKALPSTSQVTEAHTAEIRPGA
FIRQGRKLDINFGAEGNRYYEANYWQLPDGIHYNGCSEANVTKEKFVTSCINATQAVNQ
EELSREKQDNSLYQRVLWQLIRELCSNKQCDFWLERGAGLRVTLDQPMMLCLLVFIWFIVK*           

>PRND_pcw Physeter catodon sperm whale middle fragment
KALPSTSQVTEAHTAEIVPGAFIKQGRKLDIDFGAEGNRYYEANYWQFPDGIHYNGCSEA
SVTKEKFVTSCINATQEVNQEELSLEKPDNKLYQRVLWRLIRELCSI

>PRND_ssc Sus scrofa pig full length ti|470951185 
MRKHLGGRRWAIVCILLFSQLSEVKARGIKHRIKWNRKALPSTSQVTEAHTAEMRPGAFI
KQGRKLDIDFGAEGNRYYEANYWRFPDGIHYNGCSEVNVTKEKFVTSCINTTQAANQEE
SHEKPDNKLYQRVLWRLIRELCSIKHCDFWLERGAGLRVTMDQPMMLCLLVFIwfivk*  

>PRND_sar Sorex araneus shrew Laurasiatheria; Insectivora  7.23M tr
MRKHLGWCLAIACVLLLCQVPAAQARGIKHRIKWGRKAPPSTPQVTEARVAEIRPGAFI
RQGRKLNLNLGGEDKRYYDAYYWQFPDGIHYNGCSSANVTREKFISGCINATRAANQ
EELSREKHDKLYQRVLWRLVRELCSLKRCDFWSERGAGLQVTVDQLMMFCLLAFTWFIMK* 

>PRND_eeu Erinaceus europaeus 10,952,642 seq complete
MRKHLGGWWLAIVCVLFFSQLSAVKARGIKHRFKWNRKALPSTNHVTEAQVSEIRPGAFI 
RQGTKLDIDLGAEANRYYEANYWQFPDGIHYNGCSEVNVTKAKFIASCINATHSANQ
EELSREKHDKLYQRVLWRLVRELCSLKRCDFWSERGAGLQVTVDQLMMFCLLAFTWFIMK*
 
>PRND_ban Boreoeuthere ancestralis
MRKHLGAWWLAIVCILLFSHLSTVKARGIKHRIKWNRKALPSTAQVTEAHVAEIRPGAFI
RQGRKLDIDFGAEGNRYYEANYWQFPDGIHYNGCSEANVTKEMFVTSCINATQAANQEEF
SREKQDNKLHQRVLWRLIRELCSIKHCDFWLERGAGLRVTLDQPVMLCLLVFIWFMVK*

>PRND_dno Dasypus novemcinctus pseudo frag
ANYWQLPDGILYDGCAEANVTKEALVAGCVNGHTAANQAELAHERQDTLHGRVLGRLIRELCALKRCKFWPDR

>PRND_dno Dasypus novemcinctus
MRKHLGGWRLAIVCVLLSGHLSMVKARGIKHRIKWNRKAAPGAAQVTEARVAEQRPGAFV
RQGRRLDIDFGAEGNRYYEANYWQLPDGILYDGCAEANVTKEALVAGCVNATQLANQAEL
AHEGQDTLHRRVLGRLIRELCALKRCKFWPDRAAGPRLVRGAPVFGGLLLLIWLLVR*

>PRND_cho Choloepus hoffmanni sloth single trace old dasypus pseudogene, ti|563245674 
MRKHLGGWWLAAVFVLLVCHLSVAKARGIKHRIKWNRKALPSAAQVTEARIAESRPGAFI
KQGRKLDIDFGAEGNKYYEANYWQFPDGIHYDGCSEANVTKEVFVTGCINATQAANQAEF
SREKHDKLHQRVLWRLIRELCSVKHCDFWLERGGGLRLTVDQPVVSACWFHLAYRE*

>PRND_laf Loxodonta africana African elephant Afrotheria 176 aa revised/corrected
MRKHLGAWWLAIAFVLLLSHLSMVTARGIKHRIKWNRKALPNTGHVTAAQVTETRPGAFI
RHGRKLDIDFGAEGNRYYEANYWQFPDGIHYDGCSEANVTKEMFVTSCINTTQAANQEEF
SRKQDNKVYQRILWRLIRELCSVKHCDFWLDRGGGLRVSLDQPVMLCLLVFIWFMVK*  

>PRND_ema Elephas maximus Asiatic elephant Afrotheria fragment 
KALPNTGHVTAAQVTETRPGAFIRHGRKLDIDFGAEGNRYYEANYWQFPDGIHYDGCSEA
NVTKEMFVTSCINTTQAANQEEFSREKQDNKVYQRILWRLIRELCSV

>PRND_pca Procavia capensis (cape rock hyrax) Afrotheria fragment AY130772 ti|1293358170
KPVTNPAHMTAAQVSDRRLGTFIRHGRKLDIDFGAEGNRYYEANYWLFPDGIHYDGCSDT
NVTKELFVTNCINTTQAVNQEEFSREKQDNKLYQRILWRLIRELCSllglDFWPERVGGLffSLDQPVMLCLLVFIWFMLK*

>PRND_ete Echinops telfairi Madagascar lesser tenrec Afrotheria 10,323,158 seq
MRKHLGAGWLALACVLLFGHLSVVPARGIKHRIKWNRKTLPSTAHVTEARVSVTRPGAFV
RHGQKLDVDLGADGNRYYEAHYWQFPDGIHYDGCSDANVTREMLVTRCINATQAANQAEF
SAPPRDQQDSRLHQRVLWRLIRELCSAKRCDFWLEGAGASRAGLDQPPVLLGLLVLLGLTVK* 
  
>PRND_tma Trichechus manatus (Caribbean manatee) Afrotheria fragment
KPLPNTPHVTAAQVSDARPGAFIRHGRKLDIDFGAEGNRYYEANYWQFPDGIHFDGCSEA
NVTKEMFVTSCINTTQAANQEEFSREKQDNKLYQRILWRLIRELCSV

>PRND_mdo Monodelphis domestica doppel genomic revised +rassf2 -prnd -prnp
MRRHLGICWIAIFFALLFSDLSLVKAKTTRQRNKSNRKGLQTNRTNPTTVQPSEKLQGTF
IRNGRKLVIDFGEEGNSYYATHYSLFPDEIHYAGCAESNVTKEVFISNCVNATRVINKLE
PLEEQNISDIYSRILEQLIKELCALNYCEFRTGKGTGLRLSLDQYVMVYLVILTCLIVK*

>PRND_meu Macropus eugenii wallaby 
MRRHLGTWWTAIFFALLFSDLSLVKAKGTRQRNKSNRKSLQTNRVNPTTAQPSEILQGAF
IRQGRKLSIDFGEEGNSYYETHYQLFPDEIHYVGCTESNVTKDIFISNCMNATHAVNNLE
TLEEKNASDIHSRVLEQLIKELCALKYCELETETGAGLKLSLDQSVMVYLVILTCLIVK*

>PRND_oan Ornithorhynchus anatinus  platypus 42% to opposum 187 aa 4 cys in register
MMTVRRRRRSGGARWLLVFLVLLSGDLSSLQARGPRPRNKAGRKPPPSNAGPDSPAPRPPAGARGTFIRRGGRLSVDFGPEGNGYY
QANYPLLPDAIVYPDCPTANGTREAFFGDCVNATHEANRGELTAGGNASDVHVRVLLRLVEELCALRDCGPALPTGPAPRPGPPGPPAALALLTLVLLGAQ*

>PRND_aca Anolis carolinensis weak but real! scaffold_1221:78,884-117,121 syntenic, oriented like PRNP but no larger
MMQRPLVVAILLTALWSEVCLCRRVSGSANRRNKKTSTTTSAPKLQSSTTATTFQGNLCRGGQMI
DNMDLEPNDKVYYKANLKIFPDGLYYPNCSLLLQPNTTKEELVGECVNFTIASNKLNLSK
GKDLSNTKERVMWVLIHHLCANESCGQPCPLLQNSGNLHYIGQVLTVFVGLIGCSFLSAK*

>PRND_xtr Xenopus tropicalis frog 182 aa single exon 2 cDNAs odd unbalanced cysteines syntenic turtle-prnp like, no laevis
MGRQNLFSCLILLLLILYCSLSSPRRAASSKKISKTTDLSRGAKRRPKVTNSPALGDLSFRGRALNVNFNLTEESELYTANLYSFPDGLYYPR
PAHLSGAGGTDEFISGCLNTTIERNKVWISQLEDDEEGDIYMSVATQVLQFLCMENYVKPTNGAVTCTGGLWVFIGVMHFFFLFRKGD*

>PRND_aca Anolis carolinensis dna sequence
atgatgcagagacctctggtagtagccatccttttgacagccctatggagcgaggtctgtctctgtcggcgagtgagcgg
atctgcaaataggcggaataagaagacttccaccaccacaagtgctcccaaacttcaaagctcaaccacagccacaacct
tccaaggaaatctttgtcgtggtggtcaaatgattgacaatatggacctcgagccaaatgacaaagtctattacaaggca
aacctgaagattttcccagatggactttattatcccaactgttccttgcttctgcagcccaacacgaccaaagaagagct
cgttggtgaatgcgtcaatttcaccattgcatcaaataagttgaacctatctaaaggcaaggatttgagcaacacaaagg
agagggtgatgtgggtcttgattcatcacttgtgtgcaaatgagtcgtgcggtcagccttgccctttgctccaaaactct
ggaaatctccactatattggccaagtactcactgtatttgttggcctaattggttgctcttttctttctgcaaaataa

Dating doppel: missing from bird and fish genomes -- but present in marsupial, lizard and frog

The prion and doppel genes represent a local tandem duplication that has descended intact in most lineages with the two genes remaining adjacent and unpseudogenized (though both have spawned additional retroposed pseudogenes). When did this gene duplication take place, did it correlate with or facilitate an evolutionary advancement, and how have the (largely unknown) normal functions of the two genes since diverged? Retention of the second copy is somewhat puzzling in view of viability of PRNP knockouts, indeed ">double knockouts.

The weak homology between prion and doppel, below 30% identity even for the conserved core, suggests either a quite ancient duplication (lamprey era?) followed by average divergence, or alternatively, a fairly recent duplication (tetrapod era?) followed by rapid evolution on the doppel side as it acquired a auxillary function (neofunctionalized). These possibilities can be distinguished by looking at earlier diverging species that put a date on the duplication event.

If the duplication is old, then doppel should be found in various draft vertebrate genomes now available -- 34 mammals (see Genome completion status), chicken, lizard, frog, zebrafish, tetraodon, fugu, medaka, and stickleback. If gene order and orientation (synteny) are conserved, doppel will lie between PRNP and RASSF2, allowing highly diverged forms to be recognized that might give unconvincing blast matches. Of course synteny can be supplemented by conservation of internal signatures such as signal peptide, post-translationally modified arginine, disulfides, glycosylation sites, alpha helices, short beta sheet (YMLG in human), and GPI attachment. Repeat structure is trickier to use because it has been lost in all doppels and shifts from biphasic hexapeptide in frog, turtles, birds, and lizards to decapeptide (platypus), nonapeptide (marsupial), to octapeptide (placentals).

The brushtail opossum (Trichosurus vulpecular) prion gene sequence was determined in 1995. It conserves structural elements overall but presents various anomalies such as nonapeptide repeats. Its sequence can be used as query at the UCSC genome browser to find a complete second marsupial PRNP sequence from Monodelphis domestica. The two proteins have 86% sequence identity (not including repeat region), not necessarily indicating faster divergence since marsupials have had longer to diverge than placental mammals. The two 'opossums' here are not closely related, Didelphimorphia vs Diprotodontia. The second marsupial genome (Macropus eugenii) has been released unassembled to NCBI trace archives; full length PRNP and PRND sequences are readily covered there by trace blastn.

Monodelphis domestica PRND is located next to PRNP in the same orientation in a gapless region of the assembly. Within placental mammals, doppel homology is about 85% identity but this drops off to 46% relative to marsupial, indicating a relative fast-evolving protein in this lineage (but not yet a pseudogene). Indeed, with nearly PRNP gene sequences available in nearly a hundred species of placentals and the mammalian phylogenetic tree topology finally determined, clade-specific rate variations could be pursued in unusual detail, with a possible correlation with rates of doppel evolution.

Monotreme represents a still earlier divergence: PRNP and PRND can be recovered from the newly released assembly of the platypus genome, Ornithorhynchus anatinus. This species, which diverged after birds but before the Metatherian/Eutherian split, has a decapeptide x 4 repeat (rather than a bird- or turtle-like hexapeptide. However its sequence, PQGGGASWGH, is unusual for its glutamine in second position and terminal histidine (reverse of usual mammal residues). In other respects it is conventional.

The chicken genome is gap-free in this area and retains mammalian gene order and orientation (PRNP+, RASSF2-, and SCL23A-). However, no dopple gene occurs in chicken in the expected position. If prion gene duplication occurred prior to chicken divergence, then doppel decayed to an unrecognizable pseudogene, translocated to an unsequenced region, or has been deleted. No evidence for a decayed pseudogene is observed using a high-sensitivity blast of reconstructed ancestral doppel. The other bird genome underway, Taeniopygia guttata (finch), also lacks PRND.

Very recently, a lizard genome became available, Anolis carolinensis. Using specialized methods, a full length PRND gene has be located in expected syntenic position and validated by retained internal signatures despite its very weak blast score. Similarly in the diploid frog, Xenopus tropicalis, an unmistakable frog doppel gene occurs at expected position and orientation. These results have a simple parsimonious explanation: the PRND duplication predated amphibian divergence, followed by a lineage-specific deletion in the bird clade. Crocodile sequencing could refine the date of this event.

In support, lizard and frog doppel queries retrieve solely mammalian and turtle PRND genes when back-blasted against the GenBank non-redundant nucleotide division. This argues against the idea of multiple independent duplications of the prion gene in lizard and frog distinct from a later lineage-specific duplication in mammals (with birds then never having a PRND to delete).

Note the more familiar tetraploid laboratory frog, Xenopus laevis, has two actively transcribed PRNP genes that differ slightly in the repeat-like region as well at a few individual amino acids. Their sequences are given below. No sign of doppel can be found in Xenopus laevis cDNA (unsurprisingly since doppel transcription is rare); no genome project is underway. There may be two doppels in this species, depending on the relative timing of PRNP duplication and the tetraploidization event and selective pressure to maintain two copies.

The prion gene in teleost fish has radically diverged from its mammalian counterpart especially in the repeat region but remains recognizable via various conserved anchor features, residual synteny, and weak homology. Fugu has a second PRNP gene on a different chromosome (resulting from the whole genome duplication in Teleostei) plus an apparent novel tandem duplication that resembles PRNP rather than PRND. Coelocanth genome has been proposed but not begun -- that could further refine the timing of PRND duplication. As it stands, that event occurred after teleost fish divergence but before that of amphibians, about 400 million years ago. The prion gene clearly represents the ancestral form and presumably function, ie, a doppel-like ancestor did not duplicate to a second gene that become PRNP. No counterpart of PRNP can be located in the initial release of the chondrichthyian genome, Callorhynchus milii, but that will be finished to higher coverage in a few months.

Doppels are evolving noticeably faster than prions but have not become pseudogenes in any species investigated to date. (Note very recent pseudogenes that have not yet acquired internal stop codons or loss of deeply invariant residues are difficult to detect.) It must be remembered that PRNP's globular region lies within the most slowly evolving quartile of mammalian genes. As a representative example, human/dog proteins have 78% identity for PRND but 87% for PRNP. A reconstructed ancestral mammal sequence for PRND shows no sign whatsoever of converging to a PRNP-like sequence, percent identity ancester-to-ancestor remains in the mid-20's. Since this uses up 100 million years of the 400 available, doppel evolved more drastically at an earlier time, perhaps soon after the duplication event.

To further investigate the co-evolution of PRNP and PRND, it is imperative to first collect gene sequences from a phylogenetically representative sample of species. PRNP tends to be over-sampled within Artiodactylia, indeed Bovidae. Doppel remains very poorly represented at dbEST even within much-studied mammals and enriched libraries, even in testis, a tissue where it is transcribed. The most economical explanation of the data is that doppel has a narrow function with limited and specialized expression in uncommon cell types, yet one important enough for gene retention.

However a respectable number of tetrapod doppel sequences have accumulated. In addition to genome projects, van Rheede and coworkers sequenced nine doppels from important early diverging clades of placental mammals. PRND from the chimpanzee genome has special interest for evaluating known human doppel polymorphisms to determine "wildtype" human. Macaque doppel can also the 2 amino acid deletion seen in human at position 122-123, showing this event predated old world monkey divergence. In fact that event can be precisely timed to the stem lying between tarsier and new world monkey divergence and shown non-homplasic using various genome projects underway:

PRND_hsa  ANQGEFQ--KPDNKLHQQVLWRLVQELCS   Homo  sapiens  (human)
PRND_ptr  ANQGEFQ--KPDNKLHQQVLWRLVRELCS   Pan  troglodytes  (chimp)
PRND_ggo  ANQGEFH--KPDNKLHQQVLWRLVRELCS   Gorilla  gorilla  (gorilla)
PRND_ppy  ANQGEFQ--KPDNKLHQQVLWRLVQELCS   Pongo  pygmaeus  (orang_sumatran)
PRND_nle  ANQGEFQ--KPDNKLHQRVLWRLVRELCS   Nomascus  leucogenys  (gibbon)
PRND_mml  ENQGEFQ--KPDNKLHQRVLWRLVQELCS   Macaca  mulatta  (rhesus)
PRND_pha  ANQGEFQ--KPDNKLHQRVLWRLVQELCS   Papio  hamadryas  (baboon)
PRND_cja  ANQAEFQ--KPDNKLHQRVLWRLVQELCS   Callithrix  jacchus  (marmoset_nwm)
PRND_tsy  ANAAERPREARDDPLHQRVLGRLVRELCS   Tarsius  syrichta  (tarsier)
PRND_mmr  ANQAEFAREKQD-KLHERILWRLTRELCS   Microcebus  murinus  (mouse_lemur)
PRND_tbe  ANQAEFSKEKQDNKLYQRVLWRLIKELCS   Tupaia  belangeri  (tree_shrew)
PRND_ocu  ANQGEFSREKQDNKLHQRVLWRLIKELCS   Oryctolagus  cuniculus  (rabbit)
PRND_opr  ANQGEFSREKQ-NKLHQRVLWRLIKELCS   Ochotona  princeps  (pika)
PRND_dor  ANQAEFAREMQD-KFYQRVLWRLTKELCA   Dipodomys  ordii  (kangaroo_rat)
PRND_str  ANQAEFSREKQDNKLHQRVLWRLIKELCS   Spermophilus  tridecemlineatus  (ground_squirrel)
PRND_rno  ANQAEFSREKQDSKLHQRVLWRLIKEICS   Rattus  norvegicus  (rat)
PRND_mmu  ANQAEFSREKQDSKLHQRVLWRLIKEICS   Mus  musculus  (mouse)
PRND_cpo  ANQAEFSREKQDNKLHQRILWRLIKELCS   Cavia  porcellus    (guinea_pig)
PRND_sar  ANQEELSREKHD-KLYQRVLWRLVRELCS   Sorex  araneus  (shrew)
PRND_eeu  ANQEELSREKHD-KLYQRVLWRLVRELCS   Erinaceus  europaeus  (hedgehog)
PRND_cfa  ANQEELSREKQDNKLHQRVLWRLIRELCS   Canis  familiaris  (dog)
PRND_fca  ANQEELSREKQDDKLYQRVLWRLNR-ECS   Felis  catus  (cat)
PRND_tte  ENQEELSREKQDDKLHQRILWRLIRELCS   Tapirus  terrestris  (tapir)
PRND_eca  ANQEELSQEKQDDKLYQRILWRLISELCS   Equus  caballus  (horse)
PRND_mlu  ANQEELSQERPDNRLHRRILWRLVRELCS   Myotis  lucifugus  (microbat)
PRND_csp  VNQEELSQEKQDKLLHQRILWQLIRELCS   Cynopterus  sphinx  (bat)
PRND_oar  ANQEELSREKQDNKLYQRVLWQLIRELCS   Ovis  aries  (sheep)
PRND_chi  ANQEELSREKQDNKLYQRVLWQLIRELCS   Capra  hircus  (goat)
PRND_bta  ANQEELSREKQDNKLYQRVLWQLIRELCS   Bos  taurus  (cow)
PRND_ttr  VNQEELSREKQDNSLYQRVLWQLIRELCS   Tursiops  truncatus  (dolphin)
PRND_ssc  ANQEE-SHEKPDNKLYQRVLWRLIRELCS   Sus  scrofa  (pig)
PRND_pcw  VNQEELSLEKPDNKLYQRVLWRLIRELCS   Physeter  catodon  (whale)
PRND_ban  ANQEEFSREKQDNKLHQRVLWRLIRELCS   Boreoeuthere  ancestralis  (boreoeuthere)
PRND_dno  ANQAELAHERQD-TLHGRVLGRLIRELCA   Dasypus  novemcinctus  (armadillo)
PRND_cho  ANQAEFSREKHD-KLHQRVLWRLIRELCS   Choloepus  hoffmanni  (sloth)
PRND_pcp  VNQEEFSREKQDNKLYQRILWRLIRELCS   Procavia  capensis  (hyrax)
PRND_ete  ANQAEFSAPQQDSRLHQRVLWRLIRELCS   Echinops  telfairi  (tenrec)
PRND_ema  ANQEEFSREKQDNKVYQRILWRLIRELCS   Elephas  maximus  (elephant)
PRND_laf  ANQEEFSR-KQDNKVYQRILWRLIRELCS   Loxodonta  africana  (elephant)
PRND_tma  ANQEEFSREKQDNKLYQRILWRLIRELCS   Trichechus  manatus  (manatee)
PRND_mdo  INKLEPLEEQNISDIYSRILEQLIKELCA   Monodelphis  domestica  (opossum)
PRND_meu  VNNLETLEEKNASDIHSRVLEQLIKELCA   Macropus  eugenii  (wallaby)
PRND_oan  ANRGELTAGGNASDVHVRVLLRLVEELCA   Ornithorhynchus  anatinus  (platypus)
PRND_aca  SNKLNLSKGKDLSNTKERVMWVLIHHLCA   Anolis  carolinensis  (lizard)
PRND_xtr RNKVWISQLEDDEEGDIYMSVATQVLQFLCM  Xenopus  tropicalis  (frog)
           ** * .  * *. ::*::**:* :* .:    

Below 45 PRND doppel sequences from land vertebrates are provided. Most are complete and reliable, but others are fragmentary and no better than the ongoing genome project from which they were extracted (initially low coverage, perhaps a single applicable trace). The phylogenetic distribution is approximately half as extensive as for PRNP. Almost all species having an available PRND would also have an available PRNP. It would be very easy to extend PRND coverage since primer design for PCR presents no issues. All sequences consist of a single coding intron. The only rare genomic event for PRND of any phylogenetic depth is the indel shown above.

11 primates (Euarchonta)
7 glires (rodents and rabbits)
14 laurasiatheres
1 ancestral boreoeuthere
2 xenarthrans
5 aftrotheres
2 marsupial
1 platypus
0 bird
1 lizard
1 amphibian

>PRND_hsa human full length
MRKHLSWWWLATVCMLLFSHLSAVQTRGIKHRIKWNRKALPSTAQITEAQVAENRPGAFIKQGRKLDIDFGAEGNRYY
EANYWQFPDGIHYNGCSEANVTKEAFVTGCINATQAANQGEFQKPDNKLHQQVLWRLVQELCSLKHCEFWLERGAGLRVTMHQPVLLCLLALIWLTVK*

>PRND_ptr Pan chimpanzee full length
MRKHLSWWWLATVCMLLFSHLSAVQARGIKHRIKWNRKALPSTAQITEAQVAENRPGAFIKQGRKLDIDFGAEGNRYY
EANYWQFPDGIHYNGCSEANVTKEVFVTGCIHATQAANQGEF
QKPDNKLHQQVLWRLVRELCSLKHCEFWLERGAGLRVTMHQPVLLCLLAFIWLMVK*

>PRND_ggo gorilla
MRKHLSWWWLATVCMLFFSHLSAVQARGIKHRIKWNRKALPSTAQITEAQVAENRPGAFIKQGRKLDIDFGAEGNRYY
EANYWQFPDGIHYNGCSEANVTKEVFVTGCINATQAANQGEF
HKPDNKLHQQVLWRLVRELCSLKHCEWLERSAGLRVTMHQPVLLCLLAFIWLMMK*

>PRND_ppy Pongo pygmaeus orangutan shares 2 bp deletion unique to primates.
MKNHLSWWLATVCMLLFSHLSAVQARGIKHRIKWNRKALPSTAQITEAPGAENRPGALSkQGR  
QLDIDFGTEGNRYYEANYWQFPDGIHYDGCSEANVTKEVFVTGCINATQAANQGEFQ
KPDNKLHQQVLWRLVQELCSLKHCEFWLERGAGLRVTMHQPVLLCLLAFIWLMVK*

>PRND_nle Nomascus leucogenys gibbon
MRKHLSWWWLATVCMLLFSHLSAVQARGIKHRIKWNRKALPSTAQITEAQVAENRPGGFIKQGRKLDIDFGAEGNRYY
EANYWQFPDGIHYDGCSEANVTKEVFV
TGCINATQAANQGEFQKPDNKLHQRVLWRLVRELCSLKRCEFWLERGAGLRVTMHQPVLLCLLAFIWLMVK*

>PRND_pha Papio hamadryas (baboon)
        WLSRLHAAPQ HLSLVVQARGIKHRIKWNRKALPSTAQITEAQVAENPPGSFIKQGRKLNIDFGAEGNRYY
EANYWQFPDGIHYDGCSEANVTKEVFVTGC
INATQAANQGEFQKPDNKLHQRVLWRLVQELCSLKRCEFWLEGGAGLRVTMHQPVLLCLPAFVWLMVK*

>PRND_mml Macaca mulata full length from trace archives
MRQHLSRWWLATVCMLLLSHLSVVQARGIKHRIKWNRKALPSTAQITEAQVAENRPGAFIKQGR
KLNIDFGAEGNRYYEANYWQFPDGIHYDGCSEANVTKEVFVTGCINATQAENQGEFQK
PDNKLHQRVLWRLVQELCSLKRCEFWLERGAGLRVTMHQPVLLCLPAFVWLMV*

>PRND_cja Callithrix jacchus 1st new world monkey 89%  
MRKHLSGWWLATVCMLLFSHLSAVQARGIKHRIKWNRKALPSAAQITEAQVAENRPGTFIKQGRKLDINFGAEGNRYY
EANYWQLPDGIHYDGCSETNVTKEVFVTGCINATQAANQAEFQKPDNKLHQRVLWRLVQELCSLKRCEFWLERGAGFRVATHQPLLLCLLAFIWLMAK*

>PRND_tsy Tarsius syrichta tarsier 
MRKPLGGWRLAVVCVLLLGLLSAARARGIKHRFKWNRKASPSAARVTEARVAESRPGAFIRRGRKLDIDFGAEGNKYY
EANYWQFPDGIHYDGCSKANVTKELLVAGCINATHAANAAERPREARDDPLHQRVLGRLVRELCSAKHCDFWPERAAAPGTARARPGALLLVSRL

>PRND_mmr Microcebus murinus
GNRYYEANYWQFPDGIHYDGCSDANVTKEVFVASCINATVAANQAEFAREKQDKLHERILWRLTRELCSVKRCEFWLERGAGLRVAEDCPTILSLLLLLWLLLR*

>PRND_tbe Tupaia belangeri tree shrew 6.7 m tr frag
RALPSTAHITEARVAENRPGAFIRQGRKLDIDFGVEGNRYYEANYWQFPDGIHYDGCSEVNVTKEMFITSCINATQA
ANQAEFSKEKQDNKLYQRVLWRLIKELCSIKHCDFWLERGAGLRVTMHLPVMLCLLVFVWFVVK*

>PRND_mmu Mus musculus mouse full length
MKNRLGTWWVAILCMLLASHLSTVKARGIKHRFKWNRKVLPSSGGQITEARVAENRPGAF
IKQGRKLDIDFGAEGNRYYAANYWQFPDGIYYEGCSEANVTKEMLVTSCVNATQAANQAE
FSREKQDSKLHQRVLWRLIKEICSAKHCDFWLERGAALRVAVDQPAMVCLLGFVWFIVK*
 
>PRND_rno Rattus norvegicus rat full length
MKNRVGTWGLAILCLLLASHLSTVKARGIKHRFKWNRKVLPSSGQITEAQV
AENRPGAFIKQGRKLDIDFGAEGNKYYAANYWQFPDGIYYEGCSEANVTKEVLVTRCVNA
TQAANQAEFSREKQDSKLHQRVLWRLIKEICSTKHCDFWLERGAALRITVDQQAMVCLLGFIWFIVK*

>PRND_cpo Cavia porcellus (guinea pig) fragment AY130773 N-extended at traces 7,499,469 seq
MKTSLCVWALVCVLLQCHFSSVAARGIKHRIRWGRKPTPSPSQVTEARVAMTRPGAFIKEGHKLNIDFGAEGNRYYETNYWQFPDGIHYNGCSDT
NVTKEVLVTSCINATQAANQAEFSREKQDNKLHQRILWRLIKELCSA

>PRND_dor Dipodomys ordii pseudogene
ITEARYRENAQGPSSIKP--QADIDF--ERAQ*YEA*YWQFADGINYEGCSRRHVTKQMF
VSRCINVTQAANQAEFAREMQD-KFYQRVLWRLTKELCAVKQCDFWLERGEGCPVAIVNR
PAMLGLLLCMWFIL

>PRND_str Spermophilus tridecemlineatus ground squirrel complete tr
MKKHLGTWGLAVVCVLLSTHLSAIKARGIKHRIKWNRKSVPSTVQITEAQVAQNPPGAF
IKQGRKLHIDFGAEGNKYYEANYWQFPDGIYYDGCSEANVTKEVFIAKCINATQAANQAE
FSREKQDNKLHQRVLWRLIKELCSVKHCDFWLEGGAGFQLSVDQPVMLCLLVFIWLMVK*

>PRND_ocu Oryctolagus cuniculus rabbit revised complete from traces 8,968,713 sequences 75%
MRKHLGAWGLAIVCVLLFSHLPPAKARGIKHRIKWNRKALPSTAQITEAHVAENRPGTFI
RQGRKLNIDFGPEGNKYYEANYWQFPDGVHYDGCSEGNVTKEAFVAKCVNATQAANQGE
FSREKQDNKLHQRVLWRLIKELCSIKHCDFWLERGAGVQVGGEQPLVLGLLLCTWLLVK*

>PRND_opr Ochotona princeps pika complete
MRKHLGGWGLAIVCVLLFSLLPTVKARGIKHRIKWNRKALPSTAQITEARVAENRPGTFIRQGQKLSIDFGPEGNKYY
EANYWQFPDGIHYDGCSEGNVTKEVFVAKCINATQAANQGEFSREKQNKLHQRVLWRLIKELCSIKHCDFWLERGAGFPVTVGQPMVVLCLLIFTWVLVK*

>PRND_cfa Canis familiaris dog full length
MRKHLGGCWLAIVCVLLLSQLSAVEARGIKHRIKWNRKALPGTSQVTEARSAEIRPGAFI
RQGRKLDIDLGPEGNRYYEANYWQFPDGIHYNGCSEANVTKEKFVTGCINATQVANQEEL
SREKQDNKLHQRVLWRLIRELCSVKRCDFWLERGAGPRVAGAQPVLLCLLAFIWFIVK*

>PRND_fca Felis catus cat improved fragment  8,248,053 traces now 9.2
MRKHLGGCWLAIVCVLLFSQLSAVKARGIKHRIKWNRKTLPSISQVTEAHTAEIRPGAFIRQGRKLDIDLG
AEGNRYYEANYWQFPDGIHYNGCSEANVTKEKFVTSCINATQVANQEELSREKQDhKLYQRVLWRLNREALLWSAHGNFGRERG

>PRND_eca Equus caballus perissodactyl
MRKHLGGCCLAIVCVLLFSQLPVVLSRGTKHRIKWNRKALPSTAKVTEARVEEIRPGAF
IRQGRKLNINFGAEGNRYYEANYWQFPDEIHYNGCSEANVTKEKFVISCINATQEANQEE
LSQEKQDDKLYQRILWRLISELCSVKHCDFGLESGTGLRVTMDQPVLLCLLVFIWFIVK*

>PRND_tte Tapirus terrestris (Brazilian tapir) perissodactyl fragment
KALPSTSKVTEAHTAEIRPGAFIRQGRKLNIDFGAEGNRYYEANYWQFPDEIHYNGCSEA
NVTKEKFVISCINATQAENQEELSREKQDDKLHQRILWRLIRELCSV 

>PRND_mlu Myotis lucifugus traces ti|983665423 complete
MRKQLGGCCLAIVCVLLFSQLAAVQARGIKHRIKWNRKGAAPPSSSQVTEAQRVEMQLRP
GAFIKHGRKLDIDFGAEANRYYEANYWQFPDEIHYNGCSSEANVTREKFVSGCINATQAA
NQEELSQERPDNRLHRRILWRLVRELCSVKHCDFWLENGAGLWVTVHRPVMPCLLVSIWFIVK*

>PRND_pva Pteropus vampyrus frameshifty frag
MRRQLGGCWLAIVCVLLFSQLSTVKARGIKHRTKWNR PCDRVS VTESQ GAFIKQGRK

>PRND_csp Cynopterus sphinx bat  Laurasiatheria; Chiroptera fragment
KTLPSPSSHVTESQTAEMRPGAFVKQGRKLNIDFGDEGNRYYEAHYWEFPDGIYYDACSKANVTKEK
FVTSCINATQAVNQEELSQEKQDKLLHQRILWQLIRELCSV

>PRND_bta Bos taurus cow full length
MRKHLGGCWLAIVCILLFSQLCSVKARGIKHRIKWNRKVLPSTSQVTEARTAEIRPGAFI
KQGRKLDIDFGVEGNRYYEANYWQFPDGIHYNGCSKANVTKEKFITSCINATQAANQEEL
SREKQDNKLYQRVLWQLIRELCSTKHCDFWLERGAGLRVTLDQPMMLCLLVFIWFIVK*

>PRND_oar Ovis aries sheep full length
MRKHLGGCWLAIVCVLLFSQLSSVKARGIKHRIKWNRKVLPSTSQVTEAHTAEIRPGAFI
KQGRKLDINFGVEGNRYYEANYWQFPDGIHYNGCSEANVTKEKFVTSCINATQVANQEEL
SREKQDNKLYQRVLWQLIRELCSIKHCDFWLERGAGLQVTLDQPMMLCLLVFIWFIVK*

>PRND_chi Capra hircus (goat)
MRKHLGGCWLAIVCVLLFSQLSSVKARGIKHRIKWNRKVLPSTSQVTEAHTAEIRPGAFIKQGRKLDINFGV
EGNRYYEANYWQFPDGIHYNGCSEANVTKEKFVTSCINATQVANQEELSREKQDNKLYQRVLWQLIREL
CSIKHCDFWLERGAGLQVTLDQPMMLCLLVFIWFIVK*

>PRND_ama Antidorcas marsupialis (springbok) Bovidae; Antilopinae
MRKHLGGCWLAIACILLFSQLSSVKARGIKHRIKWNRKVLPSTS
QVTEAHTAEIRPGAFIKQGRKLDIDFGVEGNRYYEANYWQFPDGIHYNGCSEANVTKE
KFVTSCINATQVANQEELSREKQDNKLYQRVLWQLIRELCSIKHCDFWLERGAGLRVT
LDQPMMLCLLVFIWFIVK*

>PRND_ttr Tursiops truncatus dolphin 1 trace ti|1435196145
MRKHLGGCWLAIVCVLLFSQLSSVKARGIKHRIKWNRKALPSTSQVTEAHTAEIRPGA
FIRQGRKLDINFGAEGNRYYEANYWQLPDGIHYNGCSEANVTKEKFVTSCINATQAVNQ
EELSREKQDNSLYQRVLWQLIRELCSNKQCDFWLERGAGLRVTLDQPMMLCLLVFIWFIVK*           

>PRND_pcw Physeter catodon sperm whale middle fragment
KALPSTSQVTEAHTAEIVPGAFIKQGRKLDIDFGAEGNRYYEANYWQFPDGIHYNGCSEA
SVTKEKFVTSCINATQEVNQEELSLEKPDNKLYQRVLWRLIRELCSI

>PRND_ssc Sus scrofa pig full length ti|470951185 
MRKHLGGRRWAIVCILLFSQLSEVKARGIKHRIKWNRKALPSTSQVTEAHTAEMRPGAFI
KQGRKLDIDFGAEGNRYYEANYWRFPDGIHYNGCSEVNVTKEKFVTSCINTTQAANQEE
SHEKPDNKLYQRVLWRLIRELCSIKHCDFWLERGAGLRVTMDQPMMLCLLVFIwfivk*  

>PRND_sar Sorex araneus shrew Laurasiatheria; Insectivora  7.23M tr
MRKHLGWCLAIACVLLLCQVPAAQARGIKHRIKWGRKAPPSTPQVTEARVAEIRPGAFI
RQGRKLNLNLGGEDKRYYDAYYWQFPDGIHYNGCSSANVTREKFISGCINATRAANQ
EELSREKHDKLYQRVLWRLVRELCSLKRCDFWSERGAGLQVTVDQLMMFCLLAFTWFIMK* 

>PRND_eeu Erinaceus europaeus 10,952,642 seq complete
MRKHLGGWWLAIVCVLFFSQLSAVKARGIKHRFKWNRKALPSTNHVTEAQVSEIRPGAFI 
RQGTKLDIDLGAEANRYYEANYWQFPDGIHYNGCSEVNVTKAKFIASCINATHSANQ
EELSREKHDKLYQRVLWRLVRELCSLKRCDFWSERGAGLQVTVDQLMMFCLLAFTWFIMK*
 
>PRND_ban Boreoeuthere ancestralis
MRKHLGAWWLAIVCILLFSHLSTVKARGIKHRIKWNRKALPSTAQVTEAHVAEIRPGAFI
RQGRKLDIDFGAEGNRYYEANYWQFPDGIHYNGCSEANVTKEMFVTSCINATQAANQEEF
SREKQDNKLHQRVLWRLIRELCSIKHCDFWLERGAGLRVTLDQPVMLCLLVFIWFMVK*

>PRND_dno Dasypus novemcinctus pseudo frag
ANYWQLPDGILYDGCAEANVTKEALVAGCVNGHTAANQAELAHERQDTLHGRVLGRLIRELCALKRCKFWPDR

>PRND_dno Dasypus novemcinctus
MRKHLGGWRLAIVCVLLSGHLSMVKARGIKHRIKWNRKAAPGAAQVTEARVAEQRPGAFV
RQGRRLDIDFGAEGNRYYEANYWQLPDGILYDGCAEANVTKEALVAGCVNATQLANQAEL
AHEGQDTLHRRVLGRLIRELCALKRCKFWPDRAAGPRLVRGAPVFGGLLLLIWLLVR*

>PRND_cho Choloepus hoffmanni sloth single trace old dasypus pseudogene, ti|563245674 
MRKHLGGWWLAAVFVLLVCHLSVAKARGIKHRIKWNRKALPSAAQVTEARIAESRPGAFI
KQGRKLDIDFGAEGNKYYEANYWQFPDGIHYDGCSEANVTKEVFVTGCINATQAANQAEF
SREKHDKLHQRVLWRLIRELCSVKHCDFWLERGGGLRLTVDQPVVSACWFHLAYRE*

>PRND_laf Loxodonta africana African elephant Afrotheria 176 aa revised/corrected
MRKHLGAWWLAIAFVLLLSHLSMVTARGIKHRIKWNRKALPNTGHVTAAQVTETRPGAFI
RHGRKLDIDFGAEGNRYYEANYWQFPDGIHYDGCSEANVTKEMFVTSCINTTQAANQEEF
SRKQDNKVYQRILWRLIRELCSVKHCDFWLDRGGGLRVSLDQPVMLCLLVFIWFMVK*  

>PRND_ema Elephas maximus Asiatic elephant Afrotheria fragment 
KALPNTGHVTAAQVTETRPGAFIRHGRKLDIDFGAEGNRYYEANYWQFPDGIHYDGCSEA
NVTKEMFVTSCINTTQAANQEEFSREKQDNKVYQRILWRLIRELCSV

>PRND_pca Procavia capensis (cape rock hyrax) Afrotheria fragment AY130772 ti|1293358170
KPVTNPAHMTAAQVSDRRLGTFIRHGRKLDIDFGAEGNRYYEANYWLFPDGIHYDGCSDT
NVTKELFVTNCINTTQAVNQEEFSREKQDNKLYQRILWRLIRELCSllglDFWPERVGGLffSLDQPVMLCLLVFIWFMLK*

>PRND_ete Echinops telfairi Madagascar lesser tenrec Afrotheria 10,323,158 seq
MRKHLGAGWLALACVLLFGHLSVVPARGIKHRIKWNRKTLPSTAHVTEARVSVTRPGAFV
RHGQKLDVDLGADGNRYYEAHYWQFPDGIHYDGCSDANVTREMLVTRCINATQAANQAEF
SAPPRDQQDSRLHQRVLWRLIRELCSAKRCDFWLEGAGASRAGLDQPPVLLGLLVLLGLTVK* 
  
>PRND_tma Trichechus manatus (Caribbean manatee) Afrotheria fragment
KPLPNTPHVTAAQVSDARPGAFIRHGRKLDIDFGAEGNRYYEANYWQFPDGIHFDGCSEA
NVTKEMFVTSCINTTQAANQEEFSREKQDNKLYQRILWRLIRELCSV

>PRND_mdo Monodelphis domestica doppel genomic revised +rassf2 -prnd -prnp
MRRHLGICWIAIFFALLFSDLSLVKAKTTRQRNKSNRKGLQTNRTNPTTVQPSEKLQGTF
IRNGRKLVIDFGEEGNSYYATHYSLFPDEIHYAGCAESNVTKEVFISNCVNATRVINKLE
PLEEQNISDIYSRILEQLIKELCALNYCEFRTGKGTGLRLSLDQYVMVYLVILTCLIVK*

>PRND_meu Macropus eugenii wallaby 
MRRHLGTWWTAIFFALLFSDLSLVKAKGTRQRNKSNRKSLQTNRVNPTTAQPSEILQGAF
IRQGRKLSIDFGEEGNSYYETHYQLFPDEIHYVGCTESNVTKDIFISNCMNATHAVNNLE
TLEEKNASDIHSRVLEQLIKELCALKYCELETETGAGLKLSLDQSVMVYLVILTCLIVK*

>PRND_oan Ornithorhynchus anatinus  platypus 42% to opposum 187 aa 4 cys in register
MMTVRRRRRSGGARWLLVFLVLLSGDLSSLQARGPRPRNKAGRKPPPSNAGPDSPAPRPPAGARGTFIRRGGRLSVDFGPEGNGYY
QANYPLLPDAIVYPDCPTANGTREAFFGDCVNATHEANRGELTAGGNASDVHVRVLLRLVEELCALRDCGPALPTGPAPRPGPPGPPAALALLTLVLLGAQ*

>PRND_aca Anolis carolinensis weak but real! scaffold_1221:78,884-117,121 syntenic, oriented like PRNP but no larger
MMQRPLVVAILLTALWSEVCLCRRVSGSANRRNKKTSTTTSAPKLQSSTTATTFQGNLCRGGQMI
DNMDLEPNDKVYYKANLKIFPDGLYYPNCSLLLQPNTTKEELVGECVNFTIASNKLNLSK
GKDLSNTKERVMWVLIHHLCANESCGQPCPLLQNSGNLHYIGQVLTVFVGLIGCSFLSAK*

>PRND_xtr Xenopus tropicalis frog 182 aa single exon 2 cDNAs odd unbalanced cysteines syntenic turtle-prnp like, no laevis
MGRQNLFSCLILLLLILYCSLSSPRRAASSKKISKTTDLSRGAKRRPKVTNSPALGDLSFRGRALNVNFNLTEESELYTANLYSFPDGLYYPR
PAHLSGAGGTDEFISGCLNTTIERNKVWISQLEDDEEGDIYMSVATQVLQFLCMENYVKPTNGAVTCTGGLWVFIGVMHFFFLFRKGD*

>PRND_aca Anolis carolinensis dna sequence
atgatgcagagacctctggtagtagccatccttttgacagccctatggagcgaggtctgtctctgtcggcgagtgagcgg
atctgcaaataggcggaataagaagacttccaccaccacaagtgctcccaaacttcaaagctcaaccacagccacaacct
tccaaggaaatctttgtcgtggtggtcaaatgattgacaatatggacctcgagccaaatgacaaagtctattacaaggca
aacctgaagattttcccagatggactttattatcccaactgttccttgcttctgcagcccaacacgaccaaagaagagct
cgttggtgaatgcgtcaatttcaccattgcatcaaataagttgaacctatctaaaggcaaggatttgagcaacacaaagg
agagggtgatgtgggtcttgattcatcacttgtgtgcaaatgagtcgtgcggtcagccttgccctttgctccaaaactct
ggaaatctccactatattggccaagtactcactgtatttgttggcctaattggttgctcttttctttctgcaaaataa