Prion and Doppel Gene Annotation
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CWD: conversion of cervid PrP into protease-resistant isoforms
Human genome -- still an unfinished mess; ADRA1A is 5' prion neighbor
Cow and sheep doppel genes
Quail, pigeon prion mix-up
3'UTR regulation of mRNA
Copper-induced asparagine 107 deamidation
9x repeat not neurotoxic in cultured cells
Age-related expression of prion protein in human peripheral blood leukocytes
Insulin: the first infectious amyloidosis
Yeast amyloid fibril: second gene influences fibril initiation

Cow and sheep doppel genes

9 July 00 webmaster commentary on new GenBank doppel entries
AJ251332 . Bos taurus  351 bp  and  AJ251331 Ovis aries  351 bp
Tranulis,M., Brundtland,E. and Harbitz,I
Dept. Biochemistry, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, NORWAY 
Three cheers for this much-needed data on cow and sheep doppel genes. The existence of doppel is very inconvenient to many earlier experiments based on the oft-claimed "single copy" of the prion gene, now seen to be in error; thus internationalization of prion research and genomics projects have a very important role to play.

The cow and sheep doppels proved highly informative even though they did not quite extend to mature protein. The sequences are partial, 117 amino acids. Mouse and human doppel match bovids at about the 93/117 (79%) level; the 5 doppel sequences have 114 invariant residues or 64%.

Between sheep and cow, there are 6 amino acid differences but only 7 nucleotide changes, consistent with previous indications of rapid evolution of doppel. Such a high proportion of non-synonomous changes is often taken as indicating adaption to a modified or altered function. But hundreds of millions of years have gone by already since the tandem duplication that created the prion-doppel pair: co-evolution of doppel with its conspecific prion protein as well as a diminished role admitting degeneration yet with retention of some function (relative to the full-length copy) must also be considered.

The ancestral doppel at the time of the rodent divergence can now be reconstructed with improved confidence at 16 positions (see doppel sequence resourse page). Comparing it to the prion protein at this same depth, one might expect to find considerable convergence (higher percent identity), but no, they are barely more similar.

This suggests that the prion-doppel duplication is very old (amphioxus node?) and that an initial period of rapid change brought doppel into more or less into its current form. Over the last hundred million years, it has been changing at twice the rate of the conspecific prion proteins -- but this is not fast enough to explain its divergence from prion protein if one accepts their absence in drosophila, nematode, and yeast.

Thanks to the bovid sequences, human doppel is now seen as somewhat of a mess, the problem being localized between the inner cysteine pair, with 7 residues changed out of 19, including a two base pair deletion in the second loop, suggesting a declining functional role for this domain or surface of the doppel protein. Note how detection of such regions is hugely more efficient with bioinformatics. An earlier length discrepancy in doppel can also be resolved now: mouse has a one residue insertion; the ancestral doppel length is 178.

Which species would be most informative to sequence doppel next? The reality is that 4-5 other labs are unfortunately also sequencing sheep and cow, there being no division of labor. There could be some utility in getting full length sequences, finding alleles, testing association with scrape prion gene allelesor BSE susceptibility, working out sheep kindreds, and so on. Deer, elk, and hamster are also in the works though these cannot clarify much of the gene's history given the mammalian tree topology and the species already completed.

European hedgehog (Erinaceus europaeus) and anteater (Tamandua mexicana) would be the two best placental mammala to sequence at this point, their prion genes are needed as well. The sequence of events cannot ultimately be understood until fish sequences are known.

Human doppel provides a good example of the advantages of dynamic species selection, that is, adapting the sequencing strategy in response to real-time analysis of the sequences in hand. In this case, further primate doppel sequences, say a macaque and squirrel monkey, are needed to date the observed anomaly in human doppel, which might be primate-wide or just in great apes.

Doppel is clearly still functional in primate, rodent, and bovid lineages even though it need not be retained in all lineages. Whether this function is simply the unknown one of the globular domain of prion protein expressed in different tissues or at different times, or whether it has modulated into something distinct, or whether it interacts with the prion protein or shares third-party ligands, remain to be determined.

Quail, pigeon prion mix-up

Wed 14 Jun 2000 GenBank AF256082, AF256081
Comment (webmaster): Two new full length avian prion sequences were released by Chinese investigators on 1 Jun 00. The sequences are welcome, though the title of the paper does not suggest a reason for the study (why these species were sequenced). No leading or trailing sequences were determined though the genes are said cloned -- 5'UTR exon boundaries would be of the most interest. The quail and pigeon proteins differ at 2 residues, nYPRQPGYPHNPGY vs sYPRQPGYPHNPGY in the pre-repeat region; and after the first cysteine, CFNITVTEYSIGPAAr vs CFNITVTEYSIGPAAk. These changes seem innocuous but otherwise are conserved in all birds; both occur in pigeon.

The problem with the data is that Coturnix should be less closely related to Columba (identities 265/267) than to Gallus (but amino acid identities are 257/267) because quail and chicken are both in Galliformes (indeed, both in Phasianinae), which excludes Columbiformes. At the DNA level, identities of the alignment are 798/801 (99%) and 760/804 (94%) from quail to pigeon and chicken, respectively, also beyond the pale of statistical plausibility assuming conventional taxonomy does not need a radical revision.

Pigeon and quail also share a distinctive triplet deletion in the region corresponding to 5 repeated leucines in chicken GPI anchor. These chicken codons are all CTC leucine codons suggesting replication slippage, making the shorter leucine string of pigeon/quail more likely to be ancestral than chicken. (No other avian GPI anchors have been determined; turtle aligns poorly in this region.) Pigeon, quail, duck, and chicken carry a second synapomorphic two residue deletion of SK at position 113.

The repeat region is also distinctively chicken-like in carrying an extra 'half-repeat'. The overall length agrees with two independently determined chicken sequences, suggesting that a third independent entry (M95404) represents an allele carrying an extra full repeat.

chicken ... Aves; Neognathae; Galliformes;   Phasianidae; Phasianinae; Gallus gallus
quail   ... Aves; Neognathae; Galliformes;   Phasianidae; Phasianinae; Coturnix coturnix
pigeon  ... Aves; Neognathae; Columbiformes;  Columbidae;              Columba rupestris
duck    ... Aves; Neognathae; Anseriformes;     Anatidae;              Anas platyrhynchos

Various internal controls suggest that the sequencing data is reliable for both the new sequences; therefore the problem is most likely some confusion in taxonomy. The likliest scenario, since both sequences are most closely related to chicken (to the exclusion of duck), is that both are Galliformes, making 'pigeon' the incorrectly labelled sequence. Perhaps pigeon is a mistranslation of Chinese for a quail-like bird (say, pheasant) or by some error the pigeon is simply an allele of quail representing a different individual or population and the authors haven't yet submitted their pigeon sequence.

There is no practical way of contacting the authors since GenBank refuses to carry email addresses of entry providers. A GenBank help desk staffer did respond to a webmaster query on 21 Jun 00 saying "I will forward your message to the authors of the sequence AF256081. If they respond to me directly I will notify you."

This is a highly unsatisfactory procedure; an oft-proposed remedy is to allow other researchers to attach comments linked directly from the entry, so that everyone accessing the sequence can assess any controversy or clarifications for themselves. These sequences cannot be added to the curated prion sequence database until their status is clarified.

Update: on 12 Jul 00 the same group submitted AF283319 from duck, Anas platyrhynchos, in serious conflict with a previous sequence from the same species.

Anas 2       ............................................-.......R.......
Coturnix 1   MARLLTTCCLLALLLAACTDVALSKKGKGKPSGGGWGAGSHRQPSYPRQPGYPHNPGYPH 60
Columba  1   ............................................N............... 60
Gallus   1   ............................................................ 60
Gallus   1   ............................................................ 60
Gallus   1   ............................................................ 60
Anas     1                           .............T...............Q...... 36
Tyro alb 1                           .............T...............Q..S... 36
P.turtur 1                           .............T...............Q...... 36
P.desol  1                           .............T...............Q...... 36
Vultur   1                           .............T............S..Q...... 36
Balearic 1                           .............T...............Q...... 36
Struthio 1                                                             .. 2

Anas 2       .................HNPGYP....................................
Coturnix 61  NPGYPHNPGYPHNPGYP------QNPGYPHNPGYPGWGQGYNPSSGGSYHNQKPWKPPK- 113
Columba  61  .................------....................................- 113
Gallus   61  .................------....................................- 113
Gallus   61  .................------....................................- 113
Gallus   61  .................HNPGYP....................................- 119
Anas     37  ................------------.....W-----........N..H........- 78
Tyro alb 37  .................------H.........---.......................S 87
P.turtur 37  .................------H.....Q...---...........T...........S 87
P.desol  37  .................------H.....Q...---...........T...........S 87
Vultur   37  ................------------.....---.......................S 81
Balearic 37  ................------------.....---.......................S 81
Struthio 3   .................------H.........---.......................S 53

Anas 2       -...........................................................
Coturnix 114 -TNFKHVAGAAAAGAVVGGLGGYAMGRVMSGMSYRFDSPDEYRWWNENSARYPNRVYYRD 172
Columba  114 -........................................................... 172
Gallus   114 -...............................N.H..R.......S.............. 172
Gallus   114 -...............................N.H..R.......S.............. 172
Gallus   120 -...............................N.H..........S.............. 178
Anas     79  -...............................H.H......................... 137 
Tyro alb 88  K...............................H.........Q................. 147
P.turtur 88  K...............................H.....................Q..... 147
P.desol  88  K.......................I.......H.....................Q...P. 147
Vultur   82  K...............................H.....................Q..... 141
Balearic 82  K..L............................H.....................Q..... 141
Struthio 54  K......................V...A....H.H.............AG.......... 113

Anas 2       .............................................T...........V..
Coturnix 173 YSSPVSQDVFVADCFNITVTEYSIGPAAKKNTSEAVPAANQTDVEMENKVVTKVIREMCV 232
Columba  173 ............................R............................... 232
Gallus   173 .....P..............................A.....E................. 232
Gallus   173 .....P..............................A.R...E................. 232
Gallus   179 .....P..............................A.....E................. 238
Anas     138 .G.T..................N........G...G.G....ET...T............ 197
Tyro alb 148 .....T................N............G..V...EM...T............ 207
P.turtur 148 .R....................N........V...A......ET.L.T...........I 207
P.desol  148 .R....................N........V...A......ET.L.T...........I 207
Vultur   142 ........I.............N............GL.V...ET.L.T...........I 201
Balearic 142 ......................N........V...GA.V...ET.L.T...........I 201
Struthio 142 .....T..M                                                    122
             
Anas 2       .......................-...........
Coturnix 233 QQYREYRLASGIQLHPADTWLAV-LLLLTTLFAMH 266
Columba  233 .......................-........... 266
Gallus   233 .......................L........... 267
Gallus   233 .......................L........... 267
Gallus   239 .......................L........... 273
Anas     198 ........                            205 
Tyro alb 208 ........                            215
P.turtur 208 ........                            215
P.desol  208 ........                            215
Vultur   202 ........                            209
Balearic 202 ........                            209
Struthio 
AF256081  Columba rupestris 801 bp pigeon
AF256082 . Coturnix coturnix 801 bp common quail.
 Aves; Neognathae; Galliformes; Phasianidae; Phasianinae; Coturnix.
 Zhang,L., Wang,Q., Fan,B. and Li,N.
 Molecular Cloning of Prion Genes in Quail and Pigeon [unpublished]
 

3'UTR regulation of mRNA

Molecular and Cellular Biology, July 2000, p. 4572-4579, Vol. 20, No. 13
E. G. Mbongolo Mbella,  S. Bertrand,  G. Huez, and J.-N. Octave 
"The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3' untranslated region (3'UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA njected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3'UTR containing 8 nucleotides more than the short 3'UTR allows the recovery of an efficiency of translation similar to that of the long 3'UTR.

Moreover, the two guanine residues located at the 3' ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3'UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3'UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation."

Comment (webmaster): A comprehensive new intron site, ISIS carries information on over 170,000 introns and provides tools for finding cis- and trans-acting recipient and donor intronic sites, as well as tabulating missed alternative splices at GenBank.

3'UTR regulation is also possible in the prion and doppel genes, both of which have alternative 3'UTR splicing in at least some species. However, even without alternative splicing, there has long been a mystery over the conservation of the central region of prion gene 3'UTR, namely 992 tgcatttctttatttctgtctcataattgtcaaaaaccagaattaggtc 1040 [human, post stop codon numbering, or 27203-27251 mRNA ending 27817 in U29185] is conserved in all species. There are 75 EST matches to the special region, none of which support alternative splicing.

This stretch has no evident propensity to form a hairpin. It is feasible however to look for secondary structures in the whole 2587 bp mRNA with mfold, a mRNA secondary structure prediction tool that only recently began accepting sequences this long. (The danger of studying only a fragment is apparent.) The minimal free energy structure is shown on the left, though it does not follow that this structure is formed in vivo or indeed has any significance. Its complexity suggests that experimental deletion of the conserved region might be an interesting way to proceed. Human 3' UTR remains a unique sequence within finished and unfinished human genomic sequence.

Conservation of the central region of mammalian prion 3' UTR
human    1      tgcatttctttatttctgtctcataattgtcaaaaaccagaattaggtc 49
sheep    2125   ..............g......t..............ag.a......... 2173
cow      2248   ..............g..a..--..............a..a......... 2294
mink     1814    .........c...g.......g......a..g............     1857
hamster  1414       ......c........................gt..c......... 1458
rat      2083       ......c.................a....g.g.t........... 2127
mouse    21130      ......c............c....a......g.t........    21171

Copper-induced asparagine 107 deamidation

J. Biol. Chem., Vol. 275, Issue 25, 19121-19131, June 23, 2000
Kefeng Qin, Dun-Sheng Yang, Ying Yang, M. Azhar Chishti, Ling-Jie Meng, Hans A. Kretzschmar, Christopher M. Yip, Paul E. Fraser, and David Westaway 
While PrPC rearranges in the area of codons 104-113 to form PrPSc during prion infections, the events that initiate sporadic Creutzfeldt-Jakob disease are undefined. As Cu(II) is a putative ligand for PrPC and has been implicated in the pathogenesis of Creutzfeldt-Jakob disease and other neurodegenerative diseases, we investigated the structural effects of binding.

Incubation of brain microsomes with Cu(II) generated ~30-kDa proteinase K-resistant PrP. Cu(II) had little effect on fresh recombinant PrP23-231, but aged protein characterized by conversion of Asn-107 to Asp decreased alpha-helical content by 30%, increased beta-sheet content 100%, formed aggregates, and acquired proteinase K resistance in the presence of Cu(II). These transitions took place without need for acid pH, organic solvents, denaturants, or reducing agents. Since conversion of Asn to Asp proceeds by a spontaneous pathway involving deamidation, our data suggest that covalent variants of PrPC arising in this manner may, in concert with Cu(II), generate PrPSc-like species capable of initiating sporadic prion disease.

[Comment (webmaster): An earlier study exists on spontaneous deamidation and isomerization of Asn108 in prion peptide and protein, seemingly without the benefit of copper [BBRC 1999 Aug 11;261(3):578-83]: "The only asparagine residue, Asn108, was deamidated to aspartic acid and isoaspartic acid with a half-life of about 12 days.... The same posttranslational modifications were found in recombinant murine full-length protein. On aging, 0.8 mol of isoaspartyl residue per mole of protein was detected ... A second modification was the partial isomerization of Asp226 which is only present in rodents.

Caughey et al also considered alterations at aspartyl or asparaginyl residues in the form of D-aspartate and/or L-isoaspartate could play a role in either the formation or stabilization of PrP-res [BBRC 1998 May 29;246(3):606-8] but found little support, noting however substoichiometric amounts might yet initiate or stabilize the PrP-res in vivo.

Manganese-loaded prion also becomes proteinase resistant [EMBO J 2000 Mar 15;19(6):1180-6]. M Purdey also has speculated that a foreign cation substituting at prion protein's Cu domain could initiate TSE [Med Hypotheses. 2000 Feb;54(2):278-306]. Note too transition metal-mediated glycoxidation accelerates cross-linking of beta-amyloid peptide.

One problem is that 23 species, including almost all old world monkeys (notably the TSE-susceptible macaque) have serine at this position which obviously is not subject to deamidation aging. Note that this serine/asparagine pair is not a toggle residue but basically synapomorphic: this codon position partitions species into appropriate phylogenetic groupings. Asparagine is ancestral, being found in marsupial as well as most placental lineages, all bird species, and turtle; doppel lacks a comparable region.]

Cricetulus migratorius   ser

Camelus dromedarius      ser
Lama glama               ser

Cebus apella             ser
Theropithecus gelada     ser
Cercopithecus aethiops   ser
Cercocebus aterrimus     ser
Cercopithecus dianae     ser
Cercopithecus mona       ser
Cercopithecus neglectus  ser
Cercopithecus patas      ser
Cercocebus torquatus     ser
Colobus guereza          ser
Macaca arctoides         ser
Macaca fascicularis      ser
Macaca fuscata           ser
Macaca mulatta           ser
Macaca nemestrina        ser
Macaca sylvanus          ser
Mandrillus sphinx        ser
Papio hamadryas          ser
Presbytis francoisi      ser
o.

9x repeat not neurotoxic in cultured cells

J Neurochem 2000 Jul;75(1):72-80
Chiesa R, Harris D
Insertional and point mutations in the gene encoding the prion protein (PrP) are responsible for familial prion diseases. We have previously generated lines of Chinese hamster ovary cells that express PrP molecules carrying pathogenic mutations, and found that the mutant proteins display several biochemical properties reminiscent of PrP(Sc), the infectious isoform of PrP.

To analyze the properties and effects of mutant PrP molecules expressed in cells with a neuronal phenotype, we have constructed stably transfected lines of PC12 cells that synthesize a PrP molecule carrying a nine-octapeptide insertion. We report here that this mutant PrP acquires scrapie-like properties, including detergent insolubility, protease resistance, and resistance to phospholipase cleavage of its glycolipid anchor. A detergent-insoluble and phospholipase-resistant form of the mutant protein is also released spontaneously into conditioned medium.

These scrapie-like biochemical properties are quantitatively similar to those seen in Chinese hamster ovary cells and are not affected by differentiation of the PC12 cells into sympathetic neurons by nerve growth factor. Moreover, there is no detectable effect of mutant PrP expression on the morphology or viability of the cells in either the differentiated or undifferentiated state. These results indicate that conversion of mutant PrP into a PrP(Sc)-like form does not depend critically on the cellular context, and they suggest that mutant PrP expressed in cultured cells, even those having the phenotype of differentiated neurons, is not neurotoxic.

Differential Contribution of Superoxide Dismutase Activity by Prion Protein in Vivo

Biochem Biophys Res Commun 2000 Jun 24;273(1):136-139
Wong BS, Pan T, Liu T, Li R, Gambetti P, Sy MS 
Normal prion protein (PrP(C)) is a copper binding protein and may play a role in cellular resistance to oxidative stress. Recently, copper-bound recombinant PrP(C) has been shown to exhibit superoxide dismutase (SOD)-like activity. However, as PrP(C) affinity for copper is low in comparison to other cupro-proteins, the question remains as to whether PrP(C) could contribute SOD activity in vivo.

To unravel this enigma, we compared the SOD activity in lysates extracted from different regions of the brain from wild-type mice before and after the depletion of PrP(C). We found that removal of PrP(C) from the brain lysates reduced the levels of total SOD activity. The level of contribution to the total SOD activity was correlated to the level of PrP expressed and to the predominant form of PrP present in the specific brain region. Collectively, these results provide strong evidence that PrP(C) differentially contributes to the total SOD activity in viv

Age-related expression of the cellular prion protein in human peripheral blood leukocytes

Haematologica 2000 Jun;85(6):580-587 pdf 
Age-related expression of the cellular prion protein in human peripheral blood leukocytes.
Politopoulou G, Seebach JD, Schmugge M, Schwarz HP, Aguzzi A
CJD typically affects older patients, yet victims of new-variant Creutzfeldt-Jakob disease (nvCJD) are unusually young. Because the cellular prion protein PrPC is required for disease development, we investigated age-dependent variability in cell surface PrPC expression on various subclasses of human peripheral blood leukocytes (PBL) as a possible susceptibility factor.

Three age groups of healthy individuals (mean ages: 6, 33 and 68) were studied by two-color FACS analysis of PBL with fluorescent monoclonal antibodies to PrPC and to the lineage markers CD3, CD19, CD4, and CD8. For each subclass marker, surface PrPC levels were expressed as mean fluorescence intensity ratios (MFIR) by dividing the geometric mean of the fluorescence of each test antibody by the geometric mean of its isotype-matched control antibody. PrPC expression levels in each age and lineage group were compared using appropriate non-parametric tests.

We found significant age-related differences in PrPC expression on lymphocytes (p=0.0004). The elderly expressed significantly higher levels than children (p=0.0006) and adults (p=0.0009). PrPC expression was also significantly higher in CD3+ compared to CD19+ (p=0.0004) and in CD8+ compared to CD4+ lymphocytes (p=0.0044).

If PrPC expression on PBL were a significant susceptibility factor for nvCJD, young persons would display higher levels. Instead, the elderly expressed the highest amounts of PrPC on PBL. This argues against the hypothesis that variability in cell surface expression of PrPC in PBL contributes to the exquisite susceptibility of the young to nvCJD.

Insulin: the first infectious amyloidosis

Characterization of  oligomeric states of insulin in amyloid fibril formation
Biophys. J., Vol 79, Issue 2 [full text not yet available]
Ewan J.Nettleton, Paula Tito, Margaret Sunde, Mario Bouchard, Christopher M. Dobson and Carol V.Robinson
The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions of insulin at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are obtained. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which it is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin.

At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregate formed initially is converted to twisted, unbranched fibrils containing several protofilaments.

FT-IR spectroscopy shows that both the soluble form and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.

Insulin as an amyloid-fibril protein at sites of repeated insulin injections in a diabetic patient.

Diabetologia 1988 Mar; 31(3): 158-61)
Dische,F.E.; Wernstedt,C.; Westermark,G.T.; Westermark,P.; Pepys,M.B.; Rennie,J.A.; Gilbey,S.G.; Watkins,P.J. 
A patient with Type 1 (insulin-dependent) diabetes mellitus developed localised amyloidosis at the sites of his injections of porcine insulin. A major amyloid fibril protein was extracted and, by means of its amino acid composition and amino acid sequence, it was shown to contain intact insulin molecules. Porcine insulin is the tenth protein and the first foreign protein to be chemically identified in human amyloid fibrils.

A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin

Proteins 1997 Apr;27(4):507-16 
Brange J, Dodson GG, Edwards DJ, Holden PH, Whittingham JL
The crystal structure of despentapeptide insulin, a monomeric insulin, has been refined at 1.3 A spacing and subsequently used to predict and model the organization in the insulin fibril. The model makes use of the contacts in the densely packed despentapeptide insulin crystal, and takes into account other experimental evidence, including binding studies with Congo red. The dimensions of this model fibril correspond well with those measured experimentally, and the monomer-monomer contacts within the fibril are in accordance with the known physical chemistry of insulin fibrils. Using this model, it may be possible to predict mutations in insulin that might alleviate problems associated with fibril formation during insulin therapy.

Comment (webmaster): Why are these prominent papers establishing priority for insulin never cited in the prion literature? Insulin amyloid fibrillogenesis above crosses the species barrier from pig to human (though no recruitment is mentioned). This protein can form amyloid on its own upon injection and is not to be confused with nvCJD risk by injection of UK bovine insulin (nor with amylin or Islet amyloid polypeptide IAPP, the primary constituent of the amyloid fibrils that form in the pancreas in adult-onset diabetes).

Human genome -- still a raw unfinished mess

9 Jul 00 webmaster 
They must be joking about the human genome being finished last month. It is easily verified to still be a complete mess and completely unsuited to the tasks usually put to a finished genome. For example, one contig flanking the prion gene is still given on 10 July 00 as 54 small unordered fragments and another is in 26 pieces. It is very difficult to annotate small fragments because the number of orderings is factorial, worse than exponential.

In terms of prion research, it is not possible to determine how many copies of the prion supergene family are present (4 are expected just from two tetraploidization events), whether the prion or doppel gene has given rise to pseudogenes, or whether a newly discovered unbalanced translocation of chromosome 20p to chromosome 6 includes the prion and doppel gene.

Celera and InCyte have both made extravagant claims about proprietary data that, to the extent accessible, are testable using the doppel gene as an admittedly difficult probe. Those tests were badly flunked on 30 Jun 00 by both the TIGR Gene Indexes and the EST extension service. Neither genome annotation takes note of long-published experimental results, seeking instead to annotate ab initio by computer only. Yeast genome annotation is far superior because a team of postdocs curated some 15,000 experimental publications into the annotation. While a couple more mammalian genomes would greatly help in identification of human genes, as would longer ESTs, what is really needed is more genomics-directed experimentation.

No one is quite sure how to fix the mess at GenBank. Probably the best proposal is community-wide hand curation, where the raw GenBank entry has links to web sites of labs or groups that have adopted a gene family. Some argue that these are not peer-reviewed, but the fact is, the majority of GenBank entries are not associated with any print publication and even then are rarely scrutinized by a reviewer. In fact, the data often isn't even released until months after the peer review process has been completed. An estimated 10% of the prion sequences at GenBank are flawed, attributable to older sequencing methods, lab contamination, fraud, entry typos, sequencing obstructions or compositional anomalies, species misidentification, chromosome mislabelling, and the like. GenBank refuses to correct outright errors unless the request originates from the submitting author (who may have died or changed jobs).

The human genome is sufficiently complete, however, that the webmaster could establish ADRA1A: (alpha-adrenergic receptor) as the immediate 5' neighbor of prion gene.

By way of background, the 572 aa 7x transmembrane protein alpha-adrenergic receptor belongs to the family of G-protein coupled receptors that activate a phosphatidylinositol- calcium second messenger system. Its effect is mediated by G(Q) AND G(11) proteins. The protein has aspargine glycosylation sites at residues 65 and 82. The distal portion of the long arm of chromosome 5 contains homologous adrenergic receptors ADRB2 and ADRA1B, several million bases apart. ADRA1A is part of a very large gene family (121 blastp matches) that includes receptors such as alpha1C adrenergic, beta-1-adrenergic, 5-hydroxytryptamine 1a, beta-3-adrenergic, serotonin, beta2-adrenergic, adrenergic, alpha-2c and so on, making it completely unsuitable for synteny investigations. The nomenclature of this group of genes is totally garbled, with pharmacological effect confused with phylogenetic relationships, and broadly inconsistent use of protein names by different labs.

A 1991 BBRC paper mistakenly added a negative strand ORF as amino terminus to the ADRA1A gene (M76446: MAAALRSVMMAGYLSEWRTPTYRSTEMVQRLRMEAVQHSTSTAA); this was noticed by 1994 but never corrected -- the erroneous GenBank entry persists to this day and has percolated as established fact into many secondary genomic resources.

ADRA1A should not be confused with an unrelated but similar acronym ARIA, for acetylcholine receptor-inducing activity, which was once thought to correspond to normal prion function, based on extensive copurification observed by Harris et al. PNAS 1991 Sep 1;88(17):7664-8. This would have been a most provocative result had the proteins from two adjacent genes copurified.

The account below describes the first gene model for ADRA1A, as well as its flanking genes and direction of transcription. The amino terminal 86 residues (for the time being, exon 1) lie in the highly fragmented contig AL357040. Residues 87-371 are on a larger fragment of this piece and comprise exon 2. The known mRNA finishes nicely on another contig, AL121675 = dJ779E11, where coding exon 3 (201 aa, residues 367-572) is fused with 361 bp of 3'UTR. Exon 3 begins at 101921 of minus dJ779E11; the stop codon of exon 3 ends at position 101322 leaving 101,321 bp for a 3' linking probe.

Indeed, the ADRA1A gene is quickly extended in the telomeric direction to AL356414 and to a finished sequence containing the goliath gene and a ferritin light pseudogene in contig [NT_001989 = AL031670 = dJ681N20]. ADRA1A is also extendable in the centromeric direction to [AL121781*11 = HSJ1164C1], a 215,128 bp fragmented contig known to partially overlap the prion-doppel finished contig [NT_001001 = AL12191 + AL133396] of length 100167 + 148497 bp which is known to connect to the almost finished KIAA0168 contig.

This establishes the gene order as telomere -...- ferritin light -- goliath -- ADRA1A -- prion -- doppel -- KIAA0168 -..- centromere, as predicted by GenMap00. ADRA1A is transcribed towards the telomere. It is the nearest detectable 5' flanking gene to the prion-doppel complex. The gene density is quite low, about 40% of the genome-wide average: 5 active genes in 1.18 million bp which works out to only 15,217 genes overall. While genes can be missed, it is very unlikely that the 7-8 genes needed to bring density up to average will be found. Overlap of contigs would raise observed gene density. GDB reports 179 genes so far on all of chr 20 which is 1/50 of the whole genome.

The genomic neighborhood of the prion gene is gene-poor:
NT_001989  130263 bp  goliath, ferritin light ps
AL356414   188914 bp  too fragmented to annotate
AL121675   113168 bp  ADRA1A carboxy
AL357040   155950 bp  ADRA1A amino
AL121781   215128 bp  no gene features in large fragments psL7
NT_001001  248564 bp  prion, doppel, two ps
AL133354   130915 bp  KIAA0168
total      1,182,902 bp (needs adjustment to discount overlap)
Some oddities might be pursued upon the Sanger Centre finishing these contigs. First, there is some indication of a weak tandem ADRA1A pseudogene. Second, there is an astonishing 99% genomic match of AL121781 (or distal AL357040) to 75 kbpAL133403, a chromosome 6 unfinished contig, over almost the entire 215128 bp (note AC021974 provides a seemingly redundant further chr 20 match).

This chr 6 match does not extend into NT_001001[961567643-23148-3901] nor AL121675 whether finished or unfinished. Chromosome 6 is also being sequenced by the Sanger Centre. AL133403 is 260049 bp in 30 contigs, the largest is 48504 bp. If not some data mixup, the correspondence would have to reflect a very recent, possibly allelic, translocative duplication.

Further telomerically, the newly determined gene model for attractin, ATRN, had counterparts on chr 2 and chr 10 but not chr 6. Since the fractured part of AL357040 overlaps with two small fragments and the first 70kbp of the big HSJ1164C1 contig, some fragment ordering and assembling is feasible. This in turn affords gene prediction programs a better shot. However, in a month or two, these sequences may be finished.

CWD: conversion of cervid PrP into protease resistant isoforms

10 Jul 00 GenBank and correspondents
These two CWD studies are not on Medline yet but a set of new GenBank entries has appeared. Finding of alleles in several of the species reportedly lead to delays while these could be tested. Of interest for now is the new set of upstream splice acceptors of exon 3 which may or may not compete with cervid doppel acceptor in cervids; the downstream UTR is also of some interest for rates of change of non-coding sequence.

The second study reportedly found definite conversion of normal human prion by CWD abnormal prion. The efficiency is said to be low, but then BSE conversion of human is also inefficient. So the risk of transmission of CWD to American livestock sharing the same land and winter concentration cannot be determined, nor the risk to hunters known to have eaten contaminated deer and elk, but these are now established as non-zero.

Conversion of cervid PrP into protease resistant isoforms

Journal unknown
Raymond,G.J., Bossers,A., O'Rourke,K.I. and Caughey,B.

Molecular barrier to potential transmission of chronic wasting disease to humans, cattle, and sheep

J Wildlife Diseases July 2000 ?
Raymond,G.J., Bossers,A., Raymond,L.D., O'Rourke,K.I.O., McHolland,L.E., Bryant,P.K. III, Miller,M.W., Williams,E.S., Smits,M.A. and Caughey,B.
Antilocapra americana  AF156187      854 bp CDS 31..825 flanking on both sides
Odocoileus virginianus AF156186      739 bp 96G+138N allele, missing some of signal peptide
Odocoileus virginianus AF156185      830 bp CDS 31..801 flanking on both sides
Odocoileus virginianus AF156184      830 bp CDS 31..801 96S allele flanking on both sides
Cervus elaphus nelsoni AF156183      830 bp CDS 31..801 flanking on both sides
Cervus elaphus nelsoni AF156182      830 bp CDS 31..801 132L allele flanking on both sides

Ruminant variation in region of white-tailed deer alleles: 
-- note Dama dama also has N at 138
-- non-ruminants usually have triple glycine at 96
   96                                        138
WGQGGTHSQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRY
...S........................................................
.............................................N..............
.....S.............T........................................
.....S.....................................V................
.....S......................................T...............
.....S..........................................F...........
.....S...........................................M..........
.....S............................................R.........
.....S...............................................S......
.....S......................................................
.......G..............M.....................................
.......G.........................................T........H.
.......G.............................................S......
.......G....................................................
.......................................L....................
The cervid sequences are, surprisingly, identical in their 5' and 3' UTR. The 5' UTR is identical to cattle but differs from sheep (8 distinct entries) at one position, T-9G. Perhaps even more surprising is the conservation of 37 bp of 3' UTR of sheep, cattle, goat, mule deer, red deer, bighorn sheep, greater kudu, dog, and cat (to the extent that these were determined); Blastn with default parameters finds no match at all with human or rodent 3' UTR. This makes phylogenetic sense without explaining what happened in the human/rodent lineages.

Yeast amyloid fibril: second gene influences fibril initiation

PNAS Vol.97. Number12 June 5, 2000
Herman K. Edskes and Reed B. Wickner 
Comment (webmaster): Just as the yeast amyloidosis primary protein had no direct bearing or utility to the study of mammalian prion or other amyloidoses, the newly reported secondary gene also seems irrelevent. Neither yeast protein has any plausibly homology, however distant, to any primary or secondary gene in any mammalian cross-fibril disease. Yeast are too remote to provide a model system for any human disease of this type; in any event, the better analogy is to polyglutamine fibrillar disorders rather than prion disease.

Many people complained bitterly of tactics later used to promote Griffith's protein-only theory of prions, yet those seem dignified and restrained in comparison to the shameless misrepresentations and exploitative word games used by yeast researchers. In the webmaster's opinion, yeast 'prions' are intellectually dishonesty.

A genetically engineered artefact from the get-go with no role in yeast biology, the system still qualifies as having valid diagnostic hallmarks of an amyloidosis as defined in 1980 by GG Glenner. Unfortunately, protein chemists have found that a great many proteins can be induced to form amyloid under appropriate conditions -- from bacteriophage to crown eucaryote, cross-beta sheet is emerging as a surprising new generic protein structure. In other words, just about any yeast protein could be induced to form amyloid. There is even amyloid in omelets:

Formation and Seeding of Amyloid Fibrils from Wild-type Hen Lysozyme and a Peptide Fragment from the beta-Domain

JMB pp. 541-549 (doi:10.1006/jmbi.2000.3862)
Mark R. H. Krebs,  Deborah K. Wilkins,  Evonne W. Chung,..  Christopher M. Dobson
Wild-type hen lysozyme has been converted from its soluble native state into highly organized amyloid fibrils. In order to achieve this conversion, conditions were chosen to promote partial unfolding of the native globular fold and included heating of low-pH solutions and addition of organic solvents. Two peptides derived from the beta-sheet region of hen lysozyme were also found to form fibrils very readily. The properties and morphologies of the amyloid fibrils formed by incubation either of the protein or the peptides are similar to those produced from the group of proteins associated with clinical amyloidoses.

Fibril formation by hen lysozyme was substantially accelerated when aliquots of solutions in which fibrils of either one of the peptides or the full-length protein had previously formed were added to fresh solutions of the protein, revealing the importance of seeding in the kinetics of fibril formation. These findings support the proposition that the beta-domain is of particular significance in the formation of fibrils from the full-length protein and suggest similarities between the species giving rise to fibril formation and the intermediates formed during protein folding.

There is possible interest in the yeast system as a convenient model system for quickly screening 'universal' therapeutic compounds: ones like Congo red whose interaction is cross-fibril beta strand-based and thus independent of amino acid side chain sequences. Here however, it might make more sense in some cases to work with the actual disease protein so that the screen picks up both universal and particularized therapeutic candidates on the same search. Obviously yeast membranes are of no use in addressing blood-brain barrier considerations; rapidly dividing yeast cells do not mimic long-lived neurons.

The secondary gene discussed in the paper appears completely specific to the yeast system and by no means required for initiation (indeed, the authors posted a meeting abstract for Nov 2000 on 30 factors). While it might help disentangle initiation from propagation for this particular yeast amyloidosis, this does not imply analogous genes or processes exist for mammalian amyloid systems. If methods do not carry over to study in vivo or in vitro seed formation and propagation in humans, how is disease research advanced? Secondary genes have long been known in Alzheimer: proteases that produce elevated amounts of an amyloid-prone fragment; chaperone proteins have also been implicated in various systems. Secondary genes as therapeutic targets will lack universal structural elements and must be approached individually in each disease system. The mechanism of action of the yeast secondary protein has not yet been determined.

In considering the real prion protein, it must be remembered that it interacts with a great many other proteins during its life cycle. For starters, nascent prion protein interacts with the signal peptide processing system at the ER, disulfide isomerase, an arginine modifying enzyme, glycosylation enzymes, GPI-attaching enzyme, GPI-cleaving enzymes, various proteases, and possibly protein and copper chaperones, 'protein X', and doppel. All of the respective genes are capable of polymorphism and hence modulation of disease initiation or progression. In Alzheimer, which is far more abundant, it is possible to find kindreds with rare mutations in secondary genes; some fraction of sporadic CJD might also be familial but the rarity of disease, its late onset, and difficulty of diagnosis make a similar strategy less effective.

Finally, one point of confusion (not unique to these yeast researchers) must be addressed. There is no evidence whatsoever that any real-world case of prion disease has arisen spontaneously or de novo as a rare stochastic event of normal protein expressed at normal levels. This is but one of dozens of speculative hypotheses. Sporadic CJD means simply that the origin is unknown, though sometimes prion coding gene mutations are ruled out. Regulatory regions have never been studied in sporadic CJD; thus, for no case of sporadic CJD has even the prion gene been entirely eliminated. It is indeed possible that inbreeding of sheep in the eighteenth century led to familial scrapie that then transmitted (along with familial CJD) to humans, cattle, deer, elk, and mink through diet, contaminated environment, and secondary medical procedures, ie, all sporadic CJD has ultimately been seeded by defective protein.

Highlights of the article:

Infectious proteins (prions) can arise de novo as well as by transmission from another individual. De novo prion generation is believed responsible for most cases of Creutzfeldt-Jakob disease and for initiating the mad cow disease epidemic [Belief systems are religion. To repeat, there is no experimental support for de novo origin in sporadic CJD and it is scarcely on the table for BSE -- webmaster]. However, the cellular components needed for prion generation have not been identified in any system. The [URE3] prion of Saccharomyces cerevisiae is an infectious form of Ure2p, apparently a self-propagating amyloid. We now demonstrate a protein required for de novo prion generation. Mks1p negatively regulates Ure2p and is itself negatively regulated by the presence of ammonia and by the Ras-cAMP pathway. We find that in mks1 strains, de novo generation of the [URE3] prion is blocked, although [URE3] introduced from another strain is expressed and propagates stably. Ras2Val19 increases cAMP production and also blocks [URE3] generation. These results emphasize the distinction between prion generation and propagation, and they show that cellular regulatory mechanisms can critically affect prion generation.

Attempts to study prion initiation or generation in animal systems have met little success, in part because such events are rare, and most studies have been restricted to examination of the mechanism of propagation.

The nonchromosomal yeast genetic elements [URE3] (4) and [PSI+] (5) were identified (6) as infectious protein (prion) forms of Ure2p and Sup35p, respectively, based on their genetic properties being inconsistent with a nucleic acid replicon but exactly what one would expect of a prion. First, if a prion can be cured, it will arise again in the cured strain at some low frequency because the normal form of the protein is still present. Second, overproduction of the protein will increase the frequency of de novo prion appearance. Third, the gene for the protein is needed for prion propagation, but the phenotype of mutants in the gene will be the same as the phenotype produced by the presence of the prionbecause in both cases the normal form is deficient. In fact, [URE3] and [PSI+] satisfy these criteria as prions of Ure2p and Sup35p, respectively (6). In particular, de novo appearance of [URE3] is induced by overproduction of Ure2p (6-8) and appearance of [PSI] is induced by overproduction of Sup35p (9, 10).

Amyloid is a special form of protein characterized by filamentous morphology, high beta-sheet content, protease resistance, and yellow-green birefringence on staining with the dye Congo red (reviewed in ref. 11). Amyloid accumulation is a prominent feature of Alzheimer's disease, late-onset diabetes mellitus, CJD, and many other conditions. Biochemical and cell biological studies suggest that amyloid formation by Ure2p and Sup35p is the basis for the [URE3] and [PSI+] prions (7, 12-18). In extracts of [URE3] strains, Ure2p is partially protease resistant (7), and Ure2p-green fluorescent protein (GFP) fusion proteins are aggregated in vivo specifically in [URE3] cells (17). Ure2p can form amyloid in vitro, a reaction that is specifically promoted by the part of the Ure2p molecule responsible for prion formation in vivo (18). The pattern of protease-resistant fragments found with Ure2p amyloid formed in vitro (18) is the same as that seen in extracts of [URE3] strains (7). These results suggest that [URE3] is an infectious amyloidosis of Ure2p.

Mks1p was originally described as an inhibitor of growth whose action is blocked by activity of the Ras-cAMP pathway (27). For example, deletion of MKS1 partially relieves the effects of deficiency of cAMP, suggesting that antagonizing the negative action of Mks1p is a major task of the cAMP pathway. Recently, we reported that Mks1p is part of the nitrogen regulation cascade (28) as follows: NH3 Mks1p Ure2p Gln3p DAL5. Mks1p blocks the action of Ure2p unless it receives a signal indicating that a good nitrogen source (such as ammonia) is present. Thus, in the absence of Mks1p, DAL5 transcription is not activated on a poor nitrogen source. Furthermore, overexpression of Mks1p inhibits Ure2p action even in the presence of ammonia, but does not alter the steady-state levels of Ure2p (28).

In this paper we find that deletion of MKS1 dramatically impairs ability of Ure2p to change into the [URE3] prion form, whereas overproduction of Mks1p modestly increases the frequency of this prion change. Until now, there was no indication of a connection between the Ras-cAMP pathway and any prions. However, the fact that the Ras-cAMP pathway inhibits Mks1p (27) suggested to us that a constitutive RAS allele would likewise impair [URE3] prion generation, and we find this to be the case. ...

ttle is known about the mechanism of spontaneous formation of the mammalian transmissible spongiform encephalopathies. Although PrP can convert to amyloid and other forms rich in -sheet structure in vitro, such structures have not yet been shown to initiate the infectious process. Proteins capable of forming amyloids in vivo have been shown to form amyloid in vitro, but only after prolonged incubation of pure solutions at high concentrations. In most cases, such high concentrations do not exist in vivo, and certainly not as pure solutions. Thus, cellular (or extracellular) components other than the amyloid-forming proteins could have pronounced effects on the initiation and propagation of prions and amyloids. The study of such rare events is impossible in animal systems, but entirely feasible in yeast.

Propagation of [PSI+] is controlled by levels of Hsp104, with either increased or decreased levels of this chaperone resulting in loss of the prion (38). Other chaperones appear to modulate these effects (39, 40). Prion generation is finely controlled by the structure of the protein that becomes the prion (7, 10, 34, 41, 42). There appear to be both prion-promoting and prion-inhibiting regions of Ure2p and Sup35p, and their complex interactions are only beginning to be dissected. Overproduction of Sup45p prevents generation of [PSI+] (43). Since Sup45p normally forms a heterodimer with Sup35p, free Sup35p is probably the precursor of the [PSI+] form of the protein.

We show here that Mks1p is necessary for [URE3] to arise de novo, either from basal Ure2p levels or when Ure2p or its fragments are overproduced. Surprisingly, Mks1p is not necessary for [URE3] propagation. This description of a protein that is necessary for prion induction but not for prion propagation indicates that these two processes can be regulated in fundamentally different ways. We previously showed that the nitrogen source regulates Ure2p activity through Mks1p (28). Formally, the presence of ammonia inhibits the ability of Mks1p to inhibit Ure2p. This observation suggests the possibility that ammonia might also affect prion induction by Ure2p. However, nitrogen source does not affect [URE3] generation (ref. 8 and unpublished data). This finding indicates that these two effects of Mks1p on Ure2p are distinct. Nitrogen repression and derepression are also each compatible with [URE3] propagation (8), again distinguishing nitrogen regulation and the prion phenomenon. Since prion propagation is primarily a function of the N-terminal 80 residues of Ure2p, whereas nitrogen regulation is carried out by the Ure2p C-terminal domain (7, 34), our results suggest that Mks1p affects both parts of the Ure2p molecule. We have not detected covalent modification of Ure2p by Mks1p (Fig. 1B), but the mechanism of the Mks1p effect on Ure2p remains unclear.

In their original description of MKS1, Matsuura and Anraku (27) suggested that the Ras-cAMP pathway inhibits Mks1p activity. Our finding that [URE3] de novo generation was markedly defective in mks1 strains leads to the prediction that the Ras2Val19 mutation, by inactivating Mks1p, would likewise result in decreased frequency of [URE3] generation. The verification of this prediction indicates that cellular regulatory mechanisms outside the nitrogen control cascade can influence [URE3] prion formation. The broader implication is that any prion is likely to be affected by a range of cellular factors, some acting through a cascade of effects.

Our finding that Mks1p is necessary for generation of [URE3] could mean that an interaction of Ure2p and Mks1p is necessary for [URE3] prion formation. Alternatively, Mks1p may act through other cell components to allow conversion of Ure2p to the [URE3] prion form. Once conversion into the prion form is initiated, its propagation is not influenced by Mks1p, suggesting that the energy barrier for initiation is higher than that for propagation. It is likely that proteins interacting with PrP will similarly affect the frequency of spontaneous CJD. Serum amyloid P, basement membrane components (including glycosaminoglycans), and apolipoprotein E (apoE) are all regularly found in amyloid deposits (11). The association of the apolipoprotein E4 allele with Alzheimer's disease and the slowed development of amyloid A in apoE-knockout mice (44, 45) may reflect a relation similar to that we have found for Mks1p and the [URE3] amyloidosis of yeast.

The yeast prion [URE3] can be greatly induced by a functional mutated URE2 allele

The EMBO Journal, Vol. 19, No. 13 pp. 3215-3222, 2000
Eric Fernandez-Bellot, Elisabeth Guillemet and Christophe Cullin1 
The non-Mendelian element [URE3] of yeast is considered to be a prion form of the Ure2 protein. The [URE3] phenotype occurs at a frequency of 105 in haploid yeast strains, is reversible, and its frequency is increased by overexpressing the URE2 gene. We created a new mutant of the Ure2 protein, called H2p, which results in a 1000-fold increase in the rate of [URE3] occurrence. To date, only the overexpression of various C-terminal truncated mutants of Ure2p gives rise to a comparable level. The h2 allele is, thus, the first characterized URE2 allele that induces prion formation when expressed at a low level. By shuffling mutated and wild-type domains of URE2, we also created the first mutant Ure2 protein that is functional and induces prion formation. We demonstrate that the domains of URE2 function synergistically in cis to induce [URE3] formation, which highlights the importance of intramolecular interactions in Ure2p folding. Additionally, we show using a green fluorescent protein (GFP) fusion protein that the h2 allele exhibits numerous filiform structures that are not generated by the wild-type protein.

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