Prion Deletion and Insertion Mutants

Prion repeat region: origin of deletions and insertions
Draft 1.17 ... Last Rev. 8.26.97

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Table 1: Comparison of notations
Table 2: Comparison of micro-variants
Table 3: Frequency of various deletions
Table 4: Variation in cross-species repeats
Table 5: Repeat region consensus sequence
Table 6: Human insertions

Fig.1: Animation of slippage deletion
Fig.2: Animation of slippage insertion
Fig.3: Ambiguity of human deletions
Fig.4: DNA repeats: streak graphic
Fig.5: Squirrel monkey Rdc insert
Fig.6: Orangutan deletions
Fig.7: Bovine Rcb insertion
Fig.8: Camel deletion
Fig.9: Spider monkey Rb deletion
Fig.10: Silent vs. sense anomaly
Fig.11: Helix C hairpin

  • The animation shows how DNA slippage during replication of tandem repeats can lead to a repeat deletion. Here repeat Rd in the template slipped back to hybridize with Rc of the nascent chain, leading to a lack of Rd in the final new chain. Insertions differ only in that it is the nascent strand that slips, and the process can be iterated. Static view, all frames.
  • The animation shows how DNA slippage during replication of tandem repeats can lead to a repeat insertion. Here repeat Rd in the template slipped back to hybridize with Rc of the template chain, leading to an extra slightly modified Rc' in the final new chain. Insertions differ only in that it is the nascent strand that slips, and the process can be iterated. Static view, all frames.


    Numerous deletions and insertions of octapeptide units have been found in the repeat region of the mammalian prion gene. The basic unit of genetic change here is a 24 base pair repeat unit -- variation at a single base pair has not been observed in humans in the repeat region. These indels occur as deleterious mutations causing Creutzfeldt-Jakob Disease (CJD), as neutral polymorphisms, and as phylogenetically fixed changes across whole clades. Single module changes in orangutan, squirrel monkey, artiodactyls, and old world monkeys augment human data.

    Iterated DNA slippage during replication is proposed as a unified mechanism for the origin of most of these events. This model predicts, for any species, addtional classes of insertions and deletions while forbidding others. The model further clarifies the issues of ambiguous end points of deletions and wobble heterogeneity in long insertions.

    Parsimonious application of the model to mammalian sequence data allows reconstruction of the repeat region sequence at earlier phylogenetic branch nodes. A terminal nonamer in the ferungulate lineage suggests that long-repeat bovine prion has arisen recently from an internal single insertion event. The repeat region as a whole may have been generated by ancient expansions of an upstream domain, illuminating the relationship of marsupial and chicken repeat to eutherians. These results sharpen the prion probe sequence for distant BLAST II homology searches for the currently orphaned prion gene.


    CJD is a conformational disease: abnormal aggregation of the prion gene product can account for all aspects of the disorder.(39, 38, ref) About 15% of Creutzfeldt-Jakob Disease (CJD) is genetic, the rest is sporadic, iatrogenic, or the result of cross-species infection. CJD with genetic origin is, in roughly equal parts, ascribable to conventional point mutations and to large insertion mutations in a repeat region extending from codon 44 to codon 84. A deletion in this same region occurs in about 2% of the Caucasian population and seems to be a neutral polymorphism (5,10, 11, 15, 15).

    The prion gene from the 60-odd mammals sequenced to date contains a repeat region highly similar in structure to that of humans. At the protein level, the consensus sequence is a direct tandem repeat consisting of a nonapeptide followed by three octapepetides, denoted here Ra, Rb, Rc, and Rd, with typical repeat element, PHGGGWGQ, that can be tracked back to the metatherian-eutherian divergence. Strong sequence conservation over a long period suggests a significant function; however, no role has yet been assigned to this domain nor to the prion protein as a whole. The prion gene is an orphan, having no known homologues earlier than teleosts.(salmon )

    In fact, the role for this domain may reside mainly in nucleic acid rather than protein. The early part of the repeat domain is associated with an evolutionarily stable helix in mRNA called helix C. In other genes expressed in brain, mRNA structure has been suggested to target the site of translation or regulate its frequenccy. The repeat domain is easily cleaved by proteolysis and is seldom purified with intact protein; theoretical secondary structure prediction and NMR studies in mice indicate no fixed structure for the repeat domain. Silent mutations occur here with markedly reduced frequency compared with downstream domains that are equally well conserved at the amino acid sequence level, supporting selective bias at the nucleotide level (see Fig.9).

    A rather striking assemblege of human insertions and deletions -- always comprised of whole 24 base pair modules in the repeat region-- have accumulated from the screening of large numbers of CJD victims, family members, neurological patients, and controls for mutations in the prion gene. The larger insertions (four to nine repeats, sometimes imperfect) are causally associated with CJD, while shorter insertions (two and four) have less pronounced effects, and deletions (one module, three separate locations) are not clearly associated 5with disease. Only one homozygous deletion case is on record 3, in a 33-year old woman with a CJD-compatible dementia; on the other hand, a deleted allele may have ameliorating effects.(ref )Single inserts have not been seen in humans though they would seemingly not be lethal.

    Possibly, long-term study of individuals carrying deletions would show a very late onset CJD or carrier status, a concern for blood transfusion recipients. No (immuno)pathology has been reported for individuals carrying only this allele.

    Insertions and deletions in this same region have occured and become fixed in other species, most notably a single octamer repeat insertion in the squirrel monkey and a terminal nonamer insertion in the ferungulate lineage. (Some cattle exhibit a single repeat deletion polymorphism; this must be a derived condition because sheep, pigs, goats, ferrets, mink, oryx, camel, kudu, other cattle, deer, and elk prion retain the full complement of repeats.)

    Any proposed explanatory mechanism for these mutations must address the issues of modularity, specificity of end points, asymmetry between single deletions and insertions, wobble microheterogenity in some repeats, origin of complex large insertions, and non-observed but seemingly plausible events.

    Recombination, in the form of unequal crossing-over, has been put forward by several authors(4, 5, 17, (20 as the mechanism by which insertions and deletions arise in the prion gene, though doubts have arisen as larger and more complex insertions were discovered. A recombination mechanism predicts that single repeat deletions are accompanied by an equal incidence ofreciprocal recombinants (correponding single repeat insertions); however these have never been observed. Recombination also does not account for wobble imperfections in repeats -- this would make recombination a potent point mutagen throughout the human genome since repeats are found in many genes, whereas recombination is usually viewed as a DNA repair and gene shuffling mechanism of high fidelity. Nonetheless, unequal crossing-over is capable of producing some observed products and may be responsible for a portion of them.

    DNA slippage during replication has been proposed(23-33) for mutational change in other genes with repeat regions, as well as in an evolutionary context, (34-40) and is proposed applicable to the prion gene here. Oron-Karniet et al.(23 ) described the mechanism as:

    ... the mutation arose by slipped strand mispairing, creating a single-stranded loop, followed by DNA elongation, strand breathing and the formation of a mismatch bubble. An extensive literature search has revealed six additional deletion/insertion mutations in humans in which the inserted nucleotides come from the same DNA strand. Our model explains all six mutations, suggesting that rearrangement of a mismatch loop or bubble during DNA replication may be not uncommon.
    Asymmetry between insertions and deletions arises from unequal roles of the template and nascent DNA strand in replication. As with recombination, the basic factor giving rise to modularity is slipped hybridization of a DNA strand from a later repeat to that of an earlier one, enabled by tandem repeated sequences. The imperfect fidelity of some insertional repeats arises (in the model) not from polymerase copy error or acceptance of a wobble pair, but from incomplete 3' exonuclease editing. As will be seen, large complex insertions can arise from multiple slippage events within a single genetic epsiode, and need not be multi-generational sequential events. A single simple constraint on iterated slippage allows all observed insertions, predict further possibilities, and forbids many others.


    The current nomenclatures of the prion repeat region are unsatisfactory in three respects:

    1. Some authors number outwards from the first octamer, not counting the very similar nonamer that precedes them. This is unsatisfactory because in species such as rabbit or mouse, the nonamer has a point deletion. This notation is also confusing relative to the more popular notation that starts with the nonamer.

    2. Most authors use the numbering system of Goldfarb et al(20. This counts the repeats 5' to 3' based on human as R1 R2 R2 R3 R4. In humans, the second and third repeat are, in fact, identical at both the DNA and protein sequence level. Today, with more sequences in hand, we see that R2 R2 is not satisfactory for many species for either nucleotide or amino acid sequence; the number throws off comparisons. This system is so widely used that recalibrating here to R1 R2 R3 R4 R5 would cause considerable confusion with existing research. Because of the importance of bovine, ovine, and other TSEs, notation needs to work beyond human CJD. The notation also does not recognize partial identity in adjacent repeats, which can be adequate to support slippage(5 .

    3. Goldfarb et al (20) also established a sensible notation for extra repeats that depart from the given repeat at a single nucleotide, eg R2a, R2c, and R3g. (However other authors use R2' for R2a and R3' for R3g.(18, 19) Here, R3g differs from R3 by a T-to-G change in the third position of the seventh codon of R3. The notation is meant to imply that, say R3g, via the mechanism of its formation, is consistently a direct variant of R3. The problem here is that notation has gotten ahead of evidence of mechanism: R3g is equally one base change away from R2, or R2a is equally one base change from R3 or two from R4. (Note that interpreting R3g as an R2 variant would bring the distinct five insertional families into better agreement (21). If more variants are found, the risk of redundancy arises. The notation also has an implicit reliance on parsimony, while the differences involved are very small:

    For these reasons, the notation used here is Ra, Rb, Rc, Rd, and Re for human repeats and their eutherian homologues, in 5' to 3' order. This notation is species-independent and should be stable to new sequences since it represents the consensus ancestral sequence. Also included is Rfrag, a terminal tri-glycine repeat fragment that is best taken as part of this repeat domain. Naming of the variants should not be model-dependent, I take them here as V1, V2, V3, and V4.

    Notation used here: Other Notations
    RepeatHuman SequencePalmer(5)
    Ra cct cag ggc ggt ggt ggc tgg ggg cag (R1)
    Rb cct cat ggt ggt ggc tgg ggg cag (R2)
    Rc cct cat ggt ggt ggc tgg ggg cag (R2)
    Rd ccc cat ggt ggt ggc tgg gga cag(R3)
    Re cct cat ggt ggt ggc tgg ggt caa (R4)
    Rfrag -- -- gga ggt ggc -- -- -- -
    VariantHuman SequenceGoldfarb (20), Owen19), van Gool(18)
    V1 cct cat ggc ggt ggc tgg gga cag (R2a or R2')
    V2 cct cat ggt ggt ggc tgg ggg cag (R2c)
    V3 ccc cat ggt ggt ggc tgg gga cag (R3g or R3')
    Deletionstart and stop range*Palmer(5)
    del Acodons 54 to 82 (R2-R2, upstream of codon 76)
    del Bcodons 69 to 76(R2-R3, upstream of codon 76)
    del C codons 83 to 91 (R3-R4, downstream of codon 76)

    *Deletions can only be assigned to an ambiguity class (Fig. 4) from sequencing because of repetitive blocks.(5 )Microheterogeneity, assumption of complemetary strand pairing, and parsimony define outer boundaries here. Some authors only determined approximate sizes and positions of deletions relative to the Ncol restriction site at codon 76. The data are adequate to conclude no double deletions have been detected and that the relative frequency of occurrence of single deletions is del C >> del B > delA.

    Deletion end points in the slippage model vary according to the extent of 3' exonuclease excission during mismatch correction: the model suggests that 5' to 3' synthesis renews as soon as the last mismatch is corrected. This creates a hybrid repeat as end product: the 5' proximal part of the earlier repeat fused with the 3' end of the later repeat. The ambiguity zones for the three observed classes of human single repeat deletions, along with model-prefered end points are shown in Fig. 4 below.

    Figure 4. Deletions of 24 bp
    within an ambiguity zone
    have the same resultant sequences
    and so end points are not distinguishable.

    Ra-------------------------------Ra Rb---------------------------Rb Rc---------------------------Rc Rd-
    pro gln gly gly gly gly trp gly gln pro his gly gly gly trp gly gln pro his gly gly gly trp gly gln pro
    51  52  53  54  55  56  57  58  59  60  61  62  63  64  65  66   67 68  69  70  71  72  73  74  75  76 
    Rd---------------------------Rd Re---------------------------Re R1/2----R1/2   
    pro his gly gly gly trp gly gln pro his gly gly gly trp gly gln gly gly gly
    76  77  78  79  80  81  82  83  84  85  86  87  88  89  90  91  92  93  94

    Distinguishing deletions: Any deletion of 24 consecutive bases within an ambiguity zone produces the same final sequence. The source of DNA in the final product thus can differ, depending, in the model, on the the slipped hairpin and 3' exonuclease activity. The two extremes are shown below: the left-most and right-most start points for the deletion:

    Del A: resultant sequence after deletion of any 24 consecutive bases from codons 54 to codon 82.
    Del B: resultant sequence after deletion of any 24 consecutive bases from codons 69 to codon 76:
    Del C: resultant sequence after deletion of any 24 consecutive bases from codons 83 to codon 91:


    Frequency reported for various deletions.
    CitationDeletionAlleles*Normal allelesCommentary
    Palmer (5)del A1737careful sequencing methods, re-did N. African cases
    Perry (10) del A3121high quality SSCP sequency method S80743
    Pocchiari (6) del A9 (5)4other allele to a codon 210 CJD case
    Palmer (5)del B1737only documented case of this deletion
    Vnecak-Jones (12)del A or del B1120second deletion type found in control
    Masullo (13) del A or del B130met/met atypical dementia, homozygous deletion
    Vnecak-Jones (12, 4)del C5120Tennessee population, codon 178 proband
    Puckett (1)del C 230HeLa cell line and CDNA library, X83416
    Laplanche (2, 5 )del C35 1 family, downstream of a Ncol site
    Bosque (4, 12)del C 37in one family with D178N
    Palmer (5, 22 )del C 6737ambiguity noted of start point
    Diedrich (3)del C1 (1)6 (6) dementia cases; GenBank M81929
    Perry (10)del C 12121found in FAD + PD family, S80732
    Salvatore (7)del C2217downstream of NCol in 2 controls, no sporadics
    Reder (11)del C 13family distantly related to Bosque's family
    Brown (15)del C 2136found among 26 iatrogenic cases, 110 controls
    Brown (unpub)del C6[200]unpublished: kuru, 5 families with ataxic illness
    Windl (16)none0120UK CJD referals, allele-specific hybridization
    Owen (40)none0101methods could have detected deletions
    Goldfarb (20)none0[535]methods focused on insertions, 10 deletions expected
    Totals**all dels5319342.1-2.7% of the populations studied had some deletion**

    * Numbers in parentheses adjust for multiple family members with the same inherited deletion.
    ** Using this data at face value and assuming Hardy-Weinberg equilibrium, about one Caucasian in ten thousand is homozygous for the most comon deletion, del C. Because a single mutation may show up in distant unknown family kinships, becasue fo adoption and unknown parentage, because studies generally screened mainly neurological patients, and because controls were not always randomly selected, this and other frequencies are strictly upper limits. There is also sequencing uncertainty between del A and delB in some reports and other authors omit data on end points and only estimate the size of deletion, simply locating deletions with respect to restriction sites of NCol relative to codon 76.

    Del C is the only deletion or insertion to attain the status of common polymorphism. The issue at a population level is to decide whether, say del C, is a deletion hot spot created over and over in independent events because of the inherent proclivity of the DNA to form hairpins at the end of the repeat domain, or whether more exhaustive geneological study would show founder events are very rare but have become common alleles through neutral drift or some unknown selective bias. The occurence of similar recent deletions in other species, the unusual and lethal long insertions, and similar patterns in unrelated genes with repeads argue for an intrinsic instability in such DNA.

    Figure 1 legend: DNA repeats: streak graphic.

    DNA repeats in octapeptides of normal human prion.

    The sequence is placed on horizontal and vertical axes and compared to itself, receiving a white dot if there is a match. (This means that the diagonal is always a perfect match.) There is no new information below the diagonal because the matching process is symmetric. A moving convolution window of width 12 (four codons) removes clutter due to insignificant repeats.

    The three observed classes of deletions show up clearly on the intermittent line just above the diagonal. If slippage must be to the first adjacent region of significant complementarity, then del A, del B, and del C represent a complete set of allowable deletion classes. The extended streak for del A corresponds to the extended ambiguity zone first noted by Palmer et al. for deletion resulting in a 1234-pattern.

    Streak lines above the adjacency repeat line predict 6 additional deletion classes, corresponding to deletions to multiples of 24bp. These may be denoted as Ra/c, Rb/d, and Rc/e; Ra/d, Rb/e; and Ra/e which would result in final fusion repeats 1-3 45, 1 2-4 5, 12 3-5; 1-4 5, 1 2-5; 1-5, repectively. Slippage here must occur past the first region of possible complementarity to earlier regions; this has not been observed as yet.

    A similar graphic quickly predicts deletion patterns in any species.

    Variation in repeat regions Table 4: Variation in cross-species repeats

    Human prion repeat protein was used as a BLAST2 probe against a non-redundant set of public databases on August 11, 1997 using default settings. Homologous returned sequences (all prion orthologues) were parsed into individual repeat regions and sorted to eliminate redundancy.

    Terminology for repeat units from N to C is given in Column 1. Column 2 shows the number of species having a particular variant. Column 3 provides a proxy for species having the variant whose sequence is given in one-letter amino acid code in Column 4.

    The nonamer repeat Ra shows the most variants with eight. The sequence pqggggwgq is by far the most common and is suggested (with alternative pqgggt-wgq) to be ancestral by the marsupial sequence, pqgggtnwgq. Three point deletions occur in conjunction with various substitutions. Positions 1,2, 4, 7,8,9 are invariant. The kudu sequence tra.str is a weakly documented allele (ref ) with two surprising changes that may be attributable to cDNA error.

    The second repeat Rb is invariant as phgggwgq at the protein level in 75 species; marsupial has phpggsnwgq however. The third repeat Rc does not match Rb in three eutherian species even at the amino acid level, so this feature of the human sequence (which holds also at the DNA level) cannot be an required structural feature s. However, the slippage model, applied to an earlier era before the modern repeat structure was established, predicts ancient slippage upstream generated Ra, which in turn generated Rb-Re.

    The fourth and fifth repeats Rd, Re have minor point variation; the consensus sequence agrees with Rb and Rc. Mice and rats have an unusual serine preceding the tryptophan in both Rc and Rd.

    The fifth repeat Re has some interesting aspects. A fourth internal glycine occurs consistently in the ferrungulate clade (affecting artiodactyls, cetaceans, carnivores, and perissodactyls). This nonamer has no connection to Ra at the DNA level and probably represents a single micro-slippage event in a common ancestoral sequence.

    Certain placental mammals, most notably a bovine group, lama, giraffe, and squirrel monkey, have six repeats due to insertion events. These are similar to human insertion alleles and for alignment purposes must be considered on a case by case basis using microheterogenity in the DNA to pinpoint where the insertion occurred. Camel may have had an insertion followed by a deletion of the terminal nonamer. A single unit has been deleted in the black-handed spider monkey, Ateles geoffroyi, and five old world monkeys. Schaetztl (9 ) also reported an octamer deletion polymorphism in two of five orangutans.

    The most common allele in the bovine has six repeats; however, this is a fairly recent fixed insertion mutation. The five repeat polymorphism is the artiodactylan and mammalian norm. It may represent persistence of a more ancestral allele.

    RepeatFreq.Proxy spp.Sequence
    Ra 1 Tra.str pqeggdwgq
    Ra 2 Mus.mus pqgg-twgq
    Ra 6 Ory.cun pqggg-wgq
    Ra 35 Bos.tau pqggggwgq
    Ra 1 Cal.mol pqgggswgq
    Ra 3 Cri.gri pqgggtwgq
    Ra 1 Aot.tri pqsgg-wgq
    Ra 2 Rat.rat pqsggtwgq

    Rb 51 Tra.str phgggwgq

    Rc 48 Bos.tau phgggwgq
    Rc 2 Mus.mus phggswgq
    Rc 1 Tra.str phvggwgq

    Rd 1 Rat.rat phggg-gq
    Rd 47 Bos.tau phgggwgq
    Rd 3 Ceb.ape phggswgq

    Re 34 Pon.pyg phgggwgq
    Re 2 Rat.rat phgggwsq
    Re 7 Ate.geo -
    Re 10 Bos.tau phggggwgq




    Normal reference human sequence

    Accession P04156 D00015. (M13899 is original Kretschmar 1986 sequence, X83416 is Puckett sequence): sequence flanking repeat region:
          1          11         21         31         41         51 
      241 ILLISFLIFL IVG      
     tggctgctgg atgctggttc tctttgtggc cacatggagt gacctgggcc tctgcaagaa
     gcgcccgaag cctggaggat ggaacactgg gggcagccga tacccggggc agggcagccc
     tggaggcaac cgctacccac ctcagggcgg tggtggctgg gggcagcctc atggtggtgg
     ctgggggcag cctcatggtg gtggctgggg gcagccccat ggtggtggct ggggacagcc
     tcatggtggt ggctggggtc aaggaggtgg cacccacagt cagtggaaca agccgagtaa
     gccaaaaacc aacatgaagc acatggctgg tgctgcagca gctggggcag tggtgggggg
     ccttggcggc tacatgctgg gaagtgccat gagcaggccc atcatacatt tcggcagtga
     ctatgaggac cgttactatc gtgaaaacat gcaccgttac cccaaccaag tgtactacag
     gcccatggat gagtacagca accagaacaa ctttgtgcac gactgcgtca atatcacaat
     caagcagcac acggtcacca caaccaccaa gggggagaac ttcaccgaga ccgacgttaa
     gatgatggag cgcgtggttg agcagatgtg tatcacccag tacgagaggg aatctcaggc
     ctattaccag agaggatcga gcatggtcct cttctcctct ccacctgtga tcctcctgat
     ctctttcctc atcttcctga tagtgggatg a
    DNA for normal 123 bp or 41 aa human repeat region: nucl: 151-274 or codons 51-91
    cct cag ggc ggt ggt ggc tgg ggg cag 
    cct cat     ggt ggt ggc tgg ggg cag 
    cct cat     ggt ggt ggc tgg ggg cag 
    ccc cat     ggt ggt ggc tgg gga cag 
    cct cat     ggt ggt ggc tgg ggt caa 
                gga ggt ggc

    Repeat regions in other species: amino acid level
    camel missing gly
    cow long
    squirrel monkey long
    ate.geo;, the.gel mac.syl all short
    dwa.goa pqggg-gwg.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    ory.leu pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    Tra.str sqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq .phggggwgq	artiodactyl
    Tra.str	pqeggdwgq.phgggwgq.phvggwgq.phgggwgq          .phggggwgq	artiodactyl
    Bos.tau	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq .phggggwgq	artiodactyl
    Cam.dro	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq           	artiodactyl
    Cer.ela pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    Bos.tau	pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    Cap.hir	pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    Ovi.ari	pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    Cer.ela	pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    Odo.hem	pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    Sus.scr	pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	artiodactyl
    Mus.put	pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	carnivora
    Mus.vis	pqggggwgq.phgggwgq.phgggwgq.phgggwgq          .phggggwgq	carnivora
    rat.nor phggg-wgq.phgggwgq.phgggwgq.phgggwsq				rodent
    gol.ham phggg-wgq.phgggwgq.phgggwgq.phgggwgq				rodent
    Cri.gri	pqgggtwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq			rodent
    Cri.mig	pqgggtwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq			rodent
    Mes.aur	pqgggtwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq			rodent
    Mus.mus	pqgg-twgq.phgggwgq.phggswgq.phggswgq.phgggwgq			rodent
    Rat.nor	pqsggtwgq.phgggwgq.phgggwgq.phgggwgq.phgggwsq			rodent
    Rat.rat	pqsggtwgq.phgggwgq.phgggwgq.phggg-gq.phgggwsq			rodent
    Ory.cun	pqggg-wgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq			lagomorph
    Sai.sci	pqggg-wgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, nw
    Ate.geo	pqggg-wgq.phgggwgq.phgggwgq.phgggwgq	         monkey, nw
    Ate.pan	pqggg-wgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, nw
    Cal.jac	pqggg-wgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, nw
    Ceb.ape	pqggg-wgq.phgggwgq.phgggwgq.phggswgq.phgggwgq	 monkey, nw
    Aot.tri	pqsgg-wgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, nw
    Cal.mol	pqgggswgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Cer.ate	pqggggwgq.phgggwgq.phgggwgq.phgggwgq	         monkey, ow
    Cer.tor	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Cer.dia	pqggggwgq.phgggwgq.phgggwgq.phgggwgq	         monkey, ow
    Cer.mon	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq  monkey, ow
    Cer.neg	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq  monkey, ow
    Cer.pat	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    The.gel	pqggggwgq.phgggwgq.phgggwgq.phgggwgq	         monkey, ow
    Man.sph	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Pre.fra	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Mac.arc	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Mac.fas	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Mac.fus	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Mac.mul	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Mac.nem	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Mac.syl	pqggggwgq.phgggwgq.phgggwgq.phgggwgq	         monkey, ow
    Pap.ham	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Col.gue	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow
    Gor.gor	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow ape	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow ape
    Pan.tro	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow ape
    Hya.lar	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow ape
    Hyl.syn	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow ape
    Pon.pyg	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow ape
    Sym.syn	pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq	 monkey, ow ape

    Human Insertions

    (under development)
    Humans have not been observed to have single repeat insertions. However, inserts of length 2, 4, 5, 6, 7, 8, and 9 have been found, with longer lengths strongly associated with CJD. In some cases inserts exhibit microheterogeneity, ie, they don't exactly duplicate any of the initially existing repeats but have 1-2 silent base changes. These can vary in different insertions of the same length from unrelated families, though not within a family. They are an important clue to mechanism of insertion creation.

    Assuming contiguity of repeated stretches and parsimony based on slight sequence variations of human Rd and Re, the complex longer repeats can be interpreted in terms of wild-type repeat units. Doing so results in the table below.

    Base change differences
    -R2R3R4 R2aR2cR3g
    R2a 1 1 2 0 2 2
    R2c 1 3 3 2 0 2
    R2g 1 1 3 2 2 0

    Squirrel Monkey single repeat insertion

    Saimiri sciureu (squirrel monkey)
    Primates; Platyrrhini; Cebidae; Cebinae, Saimiri.
    The prion gene from two different animals were sequenced by different groups with accession numbers U15165 (Cervenakova) and U08310 (Schaetzl). At the protein level, the latter sequence has an extra copy of its third repeat [below] and changes of tyr to cys at codon 6 and lys to arg at codon 163. At the DNA level there are further silent changes of A to C preceding the trp codon in the penultimate repeat, and T to G at bp 606.

    Aligned squirrel monkey repeat DNA regions:

    the simplest explanation is that a tandem duplication of Rc occured; ie, Rc of the daughter strand looped out, Rd of the daughter hybridized with Rc of the template, 3' mismatches were removed, a new Rc was finished, and synthesis then continued on off Rd and Re, yielding 123345 for the newly synthesized strand.


    Pongo pygmaeus orangutan U08305

    Ambiguity zones:
    cct cag ggc ggt ggt ggc tgg ggg cag
    cct cat     ggt ggt ggc tgg ggg cag
    cct cat     ggt ggt ggc tgg ggg cag
    ccc cat     ggt ggt ggc tgg ggg cag
    cct cat     ggt ggt ggc tgg ggt caa
            gga ggt ggt 
    Signatures for distinguishable orangutan 24bp deletions:
    del A (pon.pyg) cct cat     ggt ggt ggc tgg ggg cag 
    del B (pon.pyg) ccc cat     ggt ggt ggc tgg ggt caa

    Special features of the artiodactyls

    [Under development]
    The artiodactyls present some unusual aspects in their repeat region, most notably a terminal nonamer repeat. Bovine and camel sequences have an insertion and deletion as well. Because so many artiodactyls have been sequence and because of their importance in the BSE epidemic, this whole area of the phylogenetic tree deserves special treatment. A concern of course is that certain polymorphisms might have heightened susceptibility to TSE as well as provide special dangers across species.

    X55882 is a GenBank cow sequence with 6 octapeptides; D10614 has 5 octapeptides:

    ...GG ...GG

    Ra 1 cct cag gga ggg ggt ggc tgg ggt cag 
    Rb 2 ccc cat gga     ggt ggc tgg ggc cag 
    Ri i cct cat gga     ggt ggc tgg ggc cag 
    Rc 3 cct cat gga     ggt ggc tgg ggt cag 
    Rd 4 ccc cat ggt     ggt ggc tgg gga cag 
    Rf 5 cca cat ggt ggt gga ggc tgg ggt caa 
                 ggt ggt
    Ra 1 cct cag gga ggg ggt ggc tgg ggt cag
    Rb 2 ccc cat gga     ggt ggc tgg ggc cag 
    Rc 3 cct cat gga     ggt ggc tgg ggt cag
    Rd 4 ccc cat ggt     ggt ggc tgg gga cag
    Re 5 cca cat ggt ggt gga ggc tgg ggt caa 
                 ggt ggt
    Comparing the two sequences, the simplest explanation is that (beginning with the short sequence) Rc in the nascent strand slipped back to Rb, and the insert Ri is a chimera of the proximal part of Rc with the distal part of Rb. In other words, the first codon of Ri, cct, derives from Rc whereas its last codon derives from Rb, in the slippage model.

    Y09760 camel Camelus dromedarius

    ccc cag gga ggg ggc ggc tgg ggt cag 
    ccc cac gga gga ggc     tgg ggt cag 
    ccc cac gga ggc ggc     tgg ggt caa
    ccc cac gga ggc ggc     tgg ggc cag 
    ccc cat ggt gga ggc     tgg ggt caa 
            ggt ggt ggc
    ccc cat ggt     ggt ggc tgg gga cag    cow  Rd 4 
    cca cat ggt ggt gga ggc tgg ggt caa    cow  Rf 5 
    The camel deletion is ambiguous but should be clarified by lama and any camel repeat polymorphism.  If camel originally had a terminal nonamer, then this could simply be deleted.

    U08309 black-handed spider monkey Ateles geoffroyi;
    U15164 black spider monkey x brown-headed Ateles paniscus x Ateles fusciceps
    GGG					AGG
    Ra ccc cag ggt ggt ggc tgg ggg caa 
    Rc ccc cat ggt ggc ggc tgg ggg cag 
    Rd ccc cat ggt ggc ggc tgg gga cag 
    Re cct cat ggt ggt ggc tgg ggt caa 
            gga ggt ggc
    Ra ccc cag ggt ggt ggc tgg ggg cag 
    Rb cct cat ggt ggt ggc tgg ggg caa
    Rc ccc cat ggt ggc ggc tgg ggg cag
    Rd ccc cat ggt ggc ggc tgg gga cag
    Re cct cat ggt ggt ggc tgg ggt caa 
            gca ggt ggc
    The Ateles geoffroyi results quite simply by deleting repeat Rb of the repeat region found in other species of this genus.

    The silent versus sense anomaly

    [Under development]
    The repeat region, despite the emphasis given here, has been very stable over evolutionary time at the amino acid level, even though no normal function or stable fold can be assigned to it (41, 42). A sophisticated study of secondary structure in prion mRNA has found (37) a strongly conserved stem-loop region, called helix C, just upstream and partly including Ra. The normal role is not understood but might influence transcription or translation rates or ribosomal targeting within neurons. The structure might form at least transiently or during replication, in the DNA as well and destabilize slipped strand intermediates, accounting in part for the marked instability of this region. Curiously, its length is exactly 24 bp if the bottom imperfect base pair is not included. BLASTP 2 homology searches do not find this to be a common motif in other proteins; the region is too short to search with BLASTN 2 .

    (Adapted from reference 37)

     AC CGC TAC CCA CCT CAG GGC GGT GGT -- human helix C; first 5 codons of Ra underlined 
     N   R   Y   P   P   Q   G   G   G
    If this region, or the repeat region generally, functions mainly at the nucleotide level, then third codon position could be equally as important as first or second codon position,unlike the situation at the amino acid level. This predicts that evolutionarily fixed silent mutations would be less common here than in distal regions where selection acted mainly at the protein level. The prion protein affords an opportunity to test this idea by comparison to the invariant core domain 104-122: the repeat region should be changing relatively slower at third codon position than the invariant core, if indeed the repeat experiences strong selection at the nucleotide level.


    2               GG--YNKW-KPDKP-KTNLKHVAGAAAAGAVVGGLGG  brush-tailed possum
                    .*  :.:  ** ** *:.:**:***************

    Consensus repeat region sequences

    eutherian: pqggggwgqphgggwgqphgggwgqphgggwgqphgggwgq-
    marsupial: pqgggtnwgqphpggsnwgqphpggsswgqphggsnwgq--


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    mPrP(23-231): Purification and spectroscopic characterization
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    with deletion of an alpha-helix.
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    Mice were constructed carrying prion protein (PrP) transgenes with individual regions of putative secondary structure deleted. Transgenic mice with
    amino-terminal regions deleted remained healthy at >400 days of age, whereas those with either of carboxy-terminal alpha-helices deleted spontaneously
    developed fatal CNS illnesses similar to neuronal storage diseases. Deletion of either C-terminal helix resulted in PrP accumulation within cytoplasmic
    inclusions in enlarged neurons. Deletion of the penultimate C-terminal helix resulted in proliferation of rough endoplasmic reticulum. Mice with the
    C-terminal helix deleted were affected with nerve cell loss in the hippocampus and proliferation of smooth endoplasmic reticulum. Whether children with
    the human counterpart of this malady will be found remains to be determined.
    43. Muramoto T, Scott M, Cohen FE, Prusiner SB
    Recombinant scrapie-like prion protein of 106 amino acids is soluble.
    Proc Natl Acad Sci U S A 1996 Dec 24;93(26):15457-15462
    The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of
    the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be
    deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted
    helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between
    helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues
    after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates
    solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in
    PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies
    of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies. 
     aug 8 nature:
    Based on the
                      large number of sequence-related genes encoding outer membrane proteins and the presence of
                      homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several
                      other mucosal pathogens, probably uses recombination and slipped-strand mispairing within
                      repeats as mechanisms for antigenic variation and adaptive evolution.


    I thank Mark Palmer, Katherine O'Rourke, Hermann Schaetzl, Stefan Kaluz, and Roland Heynkes for valuable cooperation.

    Supplementary Material

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