Identification of Pentatricopeptide Repeat Proteins in the ... · Identification of Pentatricopeptide Repeat Proteins in the Model Organism Dictyostelium discoideum ... (bp) Length

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2013 Article ID 586498 8 pageshttpdxdoiorg1011552013586498

Research ArticleIdentification of Pentatricopeptide Repeat Proteins inthe Model Organism Dictyostelium discoideum

Sam Manna Jessica Brewster and Christian Barth

Department of Microbiology Thomas Cherry Building La Trobe University Kingsbury Drive Bundoora VIC 3086 Australia

Correspondence should be addressed to Christian Barth cbarthlatrobeeduau

Received 18 April 2013 Accepted 11 July 2013

Academic Editor Brian Wigdahl

Copyright copy 2013 SamManna et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Pentatricopeptide repeat (PPR) proteins are RNA binding proteins with functions in organelle RNA metabolism They are foundin all eukaryotes but have been most extensively studied in plants We report on the identification of 12 PPR-encoding genes inthe genome of the protist Dictyostelium discoideum with potential homologs in other members of the same lineage and somepredicted novel functions for the encoded gene products in protists For one of the gene products we show that it localizes to themitochondria and we also demonstrate that antisense inhibition of its expression leads to slower growth a phenotype associatedwith mitochondrial dysfunction

1 IntroductionMitochondria contain their own genome and as is the casefor any other genome must maintain tight control over theexpression of their encoded gene products Mitochondrialgenes typically encode either components of the respiratorychain for ATP synthesis or the mitochondrial translationmachinery Regulating the expression of such genes is there-fore essential for normal cell function as aberrations in theregulation of mitochondrial gene expression can result indisease [1 2] Similarly to nuclear and bacterial gene expre-ssion post-transcriptional regulation is one of the mostimportant stages of mitochondrial gene expression This caninclude processing of polycistronic transcripts and liberationof structural RNAs excision of introns RNA editing andstability modifications such as polyadenylation [2]

Given that these post-transcriptional processes are highlydiverse one would expect such functions to be catalysedby many different proteins Indeed each post-transcriptionalevent often involves several proteins amongst which a largefamily of helical repeat proteins have been found to play imp-ortant roles in organelle gene expression These rather com-plex proteins are known as pentatricopeptide repeat (PPR)proteins and were originally identified during the sequencingof the genome of the model plant Arabidopsis thaliana [3]The PPR family is now known as one of the largest protein

families to exist in angiosperms with over 450 PPR-encodinggenes identified in A thaliana [4]

PPR proteins are characterised by a 35 amino acid motifoften repeated in tandem a variable number of times [35] Each PPR motif consists of two antiparallel 120572-heliceswhich interact with each other [3 5] The series of 120572-helicesform a superhelix containing a groove which can bind itsRNA ligand in a sequence-specific manner [5ndash7] Most PPRproteins function as molecular adaptors in the recruitmentof catalytic enzymes or effector proteins to target transcripts[5 7] Two classes of PPR proteins exist The P class ischaracterised by the canonical 35 amino acid motif andtypically lacks additional domains [5] The second class thePLS class consists of slightly longer and shorter PPR motifsas well as C-terminal domains such as the E E+ and DYWdomains which often have prominent roles in RNA editing[5] Indeed the presence of PLS class PPR proteins originallybelieved to be exclusive to plants correlates strongly with theoccurrence of organelle RNA editing while these proteins aretypically absent in organisms where organelle RNA editingdoes not occur [8 9] Although not as prevalent as in plantsPPR proteins are found in all eukaryotes where they havespecific roles in post-transcriptional regulation of organellegene expression Such functions include processing splicingRNA editing stabilisation polyadenylation and translational

2 International Journal of Genomics

activation [5 7] Although several of these functions areregulated by PPR proteins in plants the most commonfunction for plant PPR proteins seems to be in RNA editing aprocess which is rather common in plant organelles [5 10] Inhumans only seven PPR proteins have been identified Theyhave been shown to regulate themitochondrial transcriptomenot via RNA editing but rather through transcription andtranscript processing RNA stability polyadenylation andtranslation [11ndash15]

While the knowledge of PPR protein structure and fun-ction in non-plant organisms is expanding exponentiallylittle is known about the significance of these proteins in themitochondria of protozoa In the protists PPR proteins havebeen studied mainly in trypanosomatids where more than30 PPR genes have been identified a uniquely high numberfor a non-plant organism [16ndash19] Most of these PPR proteinsplay roles in either the stabilisation or polyadenylation ofkinetoplast transcripts and they often lack additional C-terminal domains [16ndash19] While studies into the heterolo-bosean protistNaegleria gruberi have also identified an unex-pectedly high number of PPR-encoding genes in contrast totrypanosomes a large subset of the gene products belongsto the DYW subclass of the PLS group and has thus beenimplicated in RNA editing [20 21] Despite the identificationof PPR genes in N gruberi none of their gene products havebeen functionally characterised and therefore the questionremains whether transcript stabilisation and editing are themain functions of PPR proteins in protists

Dictyostelium discoideum is a cellular slime mould bel-onging to the Amoebozoa and is a widely accepted and well-established model for studying mitochondrial genetics anddisease [22 23] Transcription of the mitochondrial genomein D discoideum has been studied in detail and some of thecore components mediating the transcription process havebeen identified InD discoideummitochondria transcriptionis initiated at a single site and the transcriptome is subjectedto several post-transcriptional modifications including pro-cessing and intron splicing as well as a single nucleotide RNAediting event that occurs in the transcript of the mitochon-drial rns gene [24ndash28] However very little is known aboutthe proteins that regulate these post-transcriptional eventsand the existence and potential role of PPR proteins in mito-chondrial RNAmetabolism have not been investigated in thisorganism Here we describe the identification of genes of thePPR protein family in D discoideum We found 12 potentialPPR proteins encoded in the D discoideum genome andsome of these proteins show significantly different featurescompared to other known PPR proteins One of the D dis-coideum proteins has been characterised in detail confirmingits mitochondrial localisationWe also demonstrate that anti-sense inhibition of its expression leads to growth defects aphenotype associatedwithmitochondrial dysfunctionWhilethe phenotypic changes resulting from antisense inhibitionof gene expression of one of these PPR proteins confirmthe importance of these proteins in mitochondrial functiontheir specific role in post-transcriptional regulation of the Ddiscoideum mitochondrial transcriptome still remains to bedetermined

2 Materials and Methods

21 Strains and Culture Conditions D discoideum strain AX2and all transformants were grown to a density of 2ndash5 times 106cellsmL in HL-5 medium at 21∘C [29 30] For non-axenicculture AX2 and all derivativeswere grownon SMplateswithKlebsiella aerogenes lawns [31] unless otherwise stated

22 Transformation of D discoideum with Vector DNA Thecalcium phosphate precipitation method was used to trans-formD discoideumwith vector DNA as described previously[32] using 20120583g of vector DNA Transformants were isolatedonMicrococcus luteus lawns on SM plates supplemented with20120583gmL G-418 [33]

23 Fluorescence Microscopy To determine the subcellularlocalisation of PtcB D discoideum transformants expressinga PtcBGFP fusion protein were analysed via fluorescencemicroscopy as described previously [34 35] Aliquots ofthe axenically grown transformant culture (sim3mL) weretransferred into a 6-well plate (BD Biosciences) containingcoverslips and the cells were allowed to settle The mediumwas removed and themitochondria were stainedwith 100 nMMitoTracker (Life Technologies) in Lo-Flo HL-5 medium for1 hour Unbound MitoTracker was removed by washing thecells four times with Lo-Flo HL-5 and twice with phosphatebuffer The cells were subsequently fixed by placing thecoverslips for 15 minutes upside down onto a 1 agarosegel in phosphate buffer containing 37 paraformaldehydeafter which the cells were washed four times with phosphatebuffered saline (PBS) Coverslips were rinsed with Milli-QsdH2O and mounted for microscopy with 90 glycerol in

PBS

24 Analysis of Growth Rates on Bacterial Lawns Growthof D discoideum cells was analysed by measuring plaqueexpansion rates on bacterial lawns as described previously[36] Briefly D discoideum cells of interest were collectedfrom the leading edge of a previously grown plaque onK aerogenes lawns The cells were then used to inoculatenormal agar plates with pregrown Escherichia coli B2 lawnsThe diameter of the plaques was measured every 8 or 16hours for 7 days to calculate the mean plaque expansion rate(mmhour) as an estimate of growth

25 Quantitative PCR The number of vector copies of theptcB antisense construct in each transformant was deter-mined using qPCR The qPCR reactions were performedusing SsoAdvanced SYBR Green Supermix (Bio-Rad) TotalgDNA extracted from each antisense transformant and fromwild type cells was used as template along with primersspecific to the cloned portion of the ptcB gene Cyclingconditions were as follows initial denaturation at 95∘C for10 minutes and then 40 cycles of denaturation at 95∘C for15 seconds followed by annealing and primer extension at60∘C for 1 minute All transcript levels were normalised tothe single copy number 120573-tubulin (tubB) gene

International Journal of Genomics 3

Table 1 Bioinformatic analysis of putative D discoideum pentatricopeptide repeat candidates The probability of the helical repeats beingpentatricopeptide repeats and the number of motifs were predicted using TPRpred and the probability of mitochondrial targeting waspredicted using Mitoprot

Gene information Protein information

Gene Chromosomelocation Gene size (bp) Length

(amino acids)Probabilityof PPR ()

Number ofPPR motifs

Probability of mitochondrialtargeting ()

ptcA 6 1518 505 9999 4 98ptcB 5 1783 528 100 9 91ptcC 5 2076 611 100 11 80ptcD 5 4321 1405 9718 7 23ptcE 1 2334 746 100 5 53ptcF 3 1596 531 100 6 93ptcG 2 1371 423 100 5 92ptcH 3 3247 1057 9637 4 88ptcI 2 3737 1163 9662 10 89ptcJ 2 3868 1258 5344 5 95ptcK 2 3623 1148 100 11 54ptcL 6 3351 1116 828 6 66

PtcA 505PtcB 528PtcC 611

1405PtcDPtcE 746tRNA methyltransferase

PtcF 531PtcG 423PtcH 1057PtcI 1163PtcJ 1258

PtcK Ubiquitin C-terminal hydrolase 2 1148PtcL 1116

Meprin and TRAF-C homology

Figure 1 Predicted domain architecture of D discoideum PPR proteins PtcA-L Blue boxes represent PPR motifs The amino acid lengthof each protein is indicated at the C-terminus of each protein Also displayed are the putative tRNA methyltransferase (yellow) MATH-like(green) and ubiquitin hydrolase-like (orange) domains of PtcE PtcJ and PtcK respectively

3 Results and Discussion

31 Identification of PPR Proteins in D discoideum We anal-ysed the D discoideum genome for any PPR-encoding genesand identified 12 gene sequences coding for putative helicalrepeat containing proteins Analysis of the protein sequencesusing the bioinformatics tool TPRpred [37] confirmed thatall candidates contained putative PPR motifs (Table 1) Thecandidates were named pentatricopeptide repeat containingproteins A-L (PtcA-L) They range in size from 423 to1405 amino acids and based on the TPRpred analysis eachcontains anywhere from 4 to 11 canonical P class PPR motifsa typical range for a non-plant PPR protein The number ofPPR proteins identified in D discoideum was also consistentwith that observed in other non-plant eukaryotes but wassignificantly less than the number of PPR proteins observedin other protists such as trypanosomatids and heteroloboseaWe did not identify any PLS class-specific features in thePPR protein candidates (Figure 1) The lack of PLS classPPR proteins in D discoideum suggests that PPR proteinsare not involved in RNA editing which correlates well with

the rather infrequent occurrence of editing in D discoideummitochondrial transcriptsThis is in contrast to plants andNgruberi which contain PLS class PPR proteins known to beinvolved in RNA editing [5 20 21]

Although most of the identified PPR proteins appear tolack any additional C-terminal domains one candidate PtcEcontains a putative C-terminal tRNA m7G46 methyltrans-ferase domain PtcE is therefore predicted to catalyse themethylation of mitochondrial tRNA species which contain aguanosine residue at position 46 a role that has not previouslybeen reported for any other PPR protein

PtcK has a putative ubiquitin carboxyl-terminal hydro-lase 2 domain However it is noteworthy that PtcK onlydisplays weak similarity to ubiquitin hydrolases and thusmay contain a non-functional domain or a similar sequenceby chance Although not homologous PtcK exhibits sim-ilarity to several members of a PPR-like family of plantorganelle RNA binding proteins (Figure 2) which contain aplant organelle RNA recognition (PORR) domain (formerlyknown as domain of unknown function 860 or DUF860)These RNA binding domains are thought to be exclusive to


NW--QFVDVYGM-DPELLSMVPR----------------------PVCAVLLLFPITEKYNW--QFVDVYGM-EPELLSMVPR----------------------PVCAVLLLFPITEKYNW--QFVDVYGM-DPELLSMVPR----------------------PVCAVLLLFPITEKYSL--GFFDVYSLDEPALLDLVPR----------------------PALALIFIAPAPMYYEW--AYFDIYSLTEPELLAFLPR----------------------PVKAIVLLFPINE--KE--ALLKNALLIEQQQLKQQQQQQQVQNQEFDNISEIQKNNNSIQIDQFAMWFPLTQLLGLPPEFRDTVCLRYPQYFRVVRMDRG-------------------PALELTHWDPELAVS

EVFRTE-------EEEKIKSQGQDVTSSVYFMKQTISNACGTIGLIHAIANNKDKMHFESEVFRTE-------EEEKIKSQGQDVTSSVYFMKQTISNACGTIGLIHAIANNKDKMHFESEVFRTE-------EEEKIKSQGQDVTSSVYFMKQTISNACGTIGLIHAIANNKDKMHFESQVRAADGT--RIAKEDGVTYRGAGPGEPVTWFRQTIGNACGLYALIHAVGNGEARTLVTE-------------DRKSSTSQQVTSSYDVIWFKQSVKNACGLYAILHSLSNNQS--LLEPLEFGQKHNNDFEIYEESLKNADQRHLHILLFYNEMVGNSELVSVIENYLERKNVYLLSSTAAELAEEESRAREAEERNLIIDRPLKFNRVRLPKGLKLTRGEARRIARFKEMPYISPYAD

GSTLKKFLEESV--SMSP-EERARYLENYDAIR---VTHETSAHEGQTEAPSIDEKVDLHGSTLKKFLEESV--SMSP-EERAKFLENYDAIR---VTHETSAHEGQTEAPSIDEKVDLHGSTLKKFLEESA--SMSP-EERARYLENYDAIR---VTHETSAHEGQTEAPNIDEKVDLHGSLLDGLLKEAE--PLRW-EARADVLYKSEELE---EAHMKAARKGDTAPPPAEERPGYHGSDLDNFLKSQS--DTSSSKNRFDDVTTDQFVLNVIKENVQTFSTGQSEAPEATADTNLHLSKLIQWYLAFDRYHLALYWLSKKISTYNSAASPILMTYFKQFSESNKNQSELVKFWNNHFSHLRSGSDEKEKHACGVVHEILSLTVEKRTLVDHLTHFREEFRFSQSLRGMIIRHPDMF

FIALVH-VDGHLYELDGR-KPFPINHG---ETSDETLLEDAIEV-CKKFMERDPDE----FIALVH-VDGHLYELDGR-KPFPINHG---KTSDETLLEDVIKV-CKKFMERDPDE----FIALVH-VDGHLYELDGR-KPFPINHG---ETSDETLLEDAIEV-CKKFMERDPDE----FIAFVKGKDGHLWELEGG-SDGPVDRGLL-EEGEDMLSEGALEKGVKKFLNYADGN----YITYVE-ENGGIFELDGRNLSGPLYLGKSDPTATDLIEQELVRVRVASYMENANEEDV--ILVYPIQKDNNNNTQQFSEDDNLIKNQNEEDGEEEQQQQVGVDESVTKIKTMTDNDKINIYVSFKG-DRDSVFLREAYKDSQLVEKNQLVLLKEKMRALVAVPRFPRRAAVGTGEEAEG-

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

6262627363536227

115115115131108596287

169169169185166656347

219219219239223716405

lowast lowast

lowast

Figure 2 Amino acid sequence alignment of PtcK from D discoideum (Dd) with ubiquitin hydrolases (UBHs) from other organismsSequences used in the alignment include UBHs from Homo sapiens (Hs accession number NP 005993) Mus musculus (Mm accessionnumber AAF64193) Bos Taurus (Bt accession number NP 001035631) Glomerella graminicola (Gg accession number EFQ25707)Saccharomyces cerevisiae (Sc accession number EDN63415) and WTF1 a PORR-containing protein from Zea mays (Zm accession numberACI96105) Only the relevant portion of the alignment is shown Boxed residues indicate conserved amino acids required for ubiquitinhydrolase activity while identical (lowast) conserved () and semiconserved () amino acids are also denoted

plants and like the domain in PtcK they not only displayweak similarity to ubiquitin hydrolases but also lack mostof the catalytic residues (Figure 2) required for such activity[38 39] Additionally the RNA binding surface of PORRproteins is similar to that of repeated helical motifs such asPPR motifs and they have been shown to mediate severalaspects of organelle gene expression at the RNA level [38 39]Only two members of this family have been characterisedand both mediate splicing of introns in organelle transcripts[38 40] Although PtcK may not be a member of this familythe features it has in common with the PORR family inaddition to the presence of PPR motifs not only imply asimilar function for PtcK in mitochondrial gene expressionbut also demonstrate a potential evolutionary link betweenPPR proteins and the PORR family In fact the latter may notbe restricted to plants as originally postulated as PtcK clearlydemonstrates that proteins similar to the PORR family existoutside of the plant lineage

Another PPR protein candidate PtcJ is predicted tocontain a meprin and TRAF-C homology (MATH) domaina domain involved in peptide cleavage and processing signaltransduction and ubiquitination [41] However given thatthese are unlikely functions for a PPR protein and that the

similarity of PtcJ to the MATH domain is weak PtcJ mayexhibit a scenario similar to PtcK in that the MATH domainis not catalytic but rather may be an RNA binding domain

Lastly TPRpred analysis of PtcL provided a low proba-bility of the candidate being a PPR protein (Table 1) despitethe fact that there were at least six PPR motifs and noother features were detected It is therefore important tonote that in previous work in Trypanosoma brucei a PPRcandidate (TbPPR9) had been identified with a TPRpredscore even lower than that obtained for PtcL but the T bruceiprotein was later shown to be a bona fide PPR protein [19]Considering this and taking into account the degeneratenature of PPR motifs it is not unreasonable to postulate thatPtcL despite its low probability score may also be a bona fidePPR protein

32 A D discoideum PPR Candidate Localizes to Mitochon-dria Additional in silico analysis of the protein sequencesindicated that most of these candidates are predicted tocontain N-terminal mitochondrial targeting signals (Table 1)as inferred by the software programMitoprot [42] Followingtheir initial identification one PPR candidate PtcB wasselected for further analysis To confirm its mitochondrial


(a) (b) (c)

Figure 3 Subcellular localisation of PtcB Fluorescencemicroscopy ofD discoideum cells (a) expressing a PtcBGFP fusion protein(b) stainedwith Mitotracker (c) indicating that the fusion protein and the mitochondria colocalise

localisation a fusion gene was created containing the 51015840 endof the ptcB gene (414 bp) encoding the putative mitochon-drial targeting signal fused to the gene encoding the greenfluorescent protein (GFP) When this construct was trans-formed and expressed in D discoideum cells the PtcBGFPfusion protein colocalised with the mitochondria (Figure 3)confirming that PtcB is indeed a mitochondrial protein andsuggesting a physiological role for the protein within thisorganelle

33 Antisense Inhibition of D discoideum PPR ExpressionResults in Slower Growth a Phenotype Associated with Mito-chondrial Dysfunction To confirm a functional role of theD discoideum PPR protein PtcB in the mitochondria theexpression of ptcB was knocked down via antisense inhibi-tionThis involved cloning a portion of the ptcB gene (414 bp)into the D discoideum expression vector pDNeo2 [43] inthe antisense orientation relative to the actin 6 promoterExpression of the ptcB gene fragment from this promoter willsynthesise an antisense RNA transcript complementary tothe endogenous ptcBmRNA sequence Upon transformationof D discoideum with vector DNA the expression vectorrandomly integrates into the genome whereby a singlefounding vector molecule will replicate at the integration sitecreating a random number of multimers [44] As a resultof this unique co-insertional replication mechanism eachD discoideum transformant contains a different number ofcopies of the antisense construct and consequently eachtransformant exhibits a different level of antisense inhibition[27] This feature allows the antisense inhibition of a gene ina dosage-dependent manner Following transformation of Ddiscoideum cells with the ptcB antisense construct 13 anti-sense transformants were isolated To establish whether PtcBhas an essential role in mitochondrial function the growthrates for these transformants were determined by growing thetransformants on bacterial lawns In D discoideum growthhas been demonstrated to be one of the first phenotypesaffected by non-functioning mitochondria and thus slowergrowth serves as an indicator of mitochondrial dysfunction[27 36] This is because mitochondrial dysfunction triggersa cascade of pathways in D discoideum that favour the

035

04

045

05

055

06

065

07

0 50 100 150 200 250 300 350

Plaq

ue ex

pans

ion

rate

(mm

hr)

Antisense vector copy number

R2= 07301

Figure 4 Plaque expansion rates of ptcB antisense transformants onEscherichia coli B2 lawns Plaque expansion rates for ptcB antisensetransformants are plotted against the copy number of the antisenseconstruct present in each transformant a reflection of the levelof antisense inhibition The number of copies of the antisenseconstruct in each transformant was determined using qPCR Alltransformants are shaded in grey while the wild type parental strainis in black

repression of ATP consuming processes such as growth [2736] Antisense inhibition of ptcB resulted in slower plaqueexpansion rates on bacterial lawns and the severity of thisphenotype correlated with the level of antisense inhibitionof ptcB as indicated by the number of antisense constructspresent in each of the transformants (Figure 4) The slowergrowth of D discoideum antisense transformants confirmsthe important role PPR proteins play in D discoideummitochondrial functionDelayed growth upon knockdownofPPR-encoding genes has also been observed in trypanosomes[18 19] and in plants PPR mutants are known to displayphenotypes associated with chloroplast or mitochondrialdysfunction including cytoplasmic male sterility negativeeffects on embryonic development and defective photosyn-thetic ability [5 45 46]

34 D discoideum PPR Proteins Possess Homologs in theCellular Slime Mould Lineage To gain further insight intothe evolution of PPR proteins in the cellular slime mould


Table 2 Putative homologs of D discoideum PPR proteins in othercellular slime moulds The presence of a homolog is noted bythe NCBI protein accession number while the absence of a clearhomolog is denoted by ldquomdashrdquo Also indicated in the parentheses arethe levels of amino acid identitysimilarity () respectively for eachprotein compared to the D discoideum homolog as determined byend to end pairwise alignments

D discoideumprotein

D purpureumhomolog

P pallidumhomolog

D fasciculatumhomolog

PtcA XP 003289503(265436) mdash mdash

PtcB XP 003288427(529712)

EFA79424(16227)

EGG14329(321494)

PtcC XP 003290170(481666)

EFA76720(374555)

EGG22645(393621)

PtcD XP 003284803(217331) mdash mdash

PtcE XP 003288663(675785)

EFA82229(463609)

EGG13534(175246)

PtcF XP 003294037(499648)

EFA79525(275503)

EGG15096(2949)

PtcG XP 003284179(619751)

EFA75260(28237)

EGG14213(495648)

PtcH XP 003286839(246412) mdash mdash

PtcI XP 003285976(244415) mdash mdash

PtcJ XP 003291714(256445) mdash mdash

PtcK XP 003293255(272443) mdash mdash

PtcL XP 003286762(258422) mdash mdash

lineage we searched for PPR protein-encoding genes in thegenomes of three other cellular slime moulds DictyosteliumpurpureumDictyostelium fasciculatum andPolysphondyliumpallidum Interestingly the search led to the identification ofwhat seemed to be homologs of most of the PPR proteinspreviously identified in D discoideum (Table 2) For most ofthese homologs it was confirmed by TPRpred analysis thatthey contain PPR motifs (Table 3) In two of the proteinshowever PPRmotifs could not be detected (protein accessionnumbers XP 003284803 and XP 003286762) despite the factthat each of the candidates displayed a high level of homologyto a specific D discoideum PPR protein (Table 2) The failureto identify any PPR motifs within these proteins may be aresult of weak conservation of their PPR motifs

None of the identified PPR proteins seem to have homo-logs in organisms outside of the cellular slime mould lineage(data not shown) A similar pattern of high conservation ofPPR homologs has also been observed previously for non-plant PPR proteins in closely related species [17 19]The highlevel of conservation not only demonstrates the importanceof PPR proteins in mitochondrial function but also suggestsa specific role for each of these homologs It is therefore likelythat these proteins fulfil more similar functions required by

Table 3 Bioinformatic analysis of D discoideum PPR proteinhomologs in other cellular slime moulds The probability of helicalrepeats being PPR and the predicted number of motifs weredetermined using TPRpred

Organism NCBI proteinaccession number

PPRprobability ()

Number ofPPR motifs

D purpureum XP 003289503 100 9D purpureum XP 003288427 100 9D purpureum XP 003290170 100 12D purpureum XP 003284803 0 0D purpureum XP 003288663 100 5D purpureum XP 003294037 100 6D purpureum XP 003284179 100 5D purpureum XP 003286839 100 9D purpureum XP 003285976 100 15D purpureum XP 003291714 089 3D purpureum XP 003293255 9996 9D purpureum XP 003286762 0 0P pallidum EFA79424 100 9P pallidum EFA76720 100 13P pallidum EFA82229 100 3P pallidum EFA79525 100 8P pallidum EFA75260 100 5D fasciculatum EGG14329 100 9D fasciculatum EGG22645 100 12D fasciculatum EGG13534 9720 3D fasciculatum EGG15096 100 8D fasciculatum EGG14213 100 6

Table 4 Bioinformatic analysis of unique PPR proteins found inone but not in other cellular slime moulds The probability ofhelical repeats being PPR and the predicted number of motifs weredetermined using TPRpred


PPRprobability ()

Number ofPPR motifs

D purpureum XP 003291713 9955 8P pallidum EFA82227 6434 3P pallidum EFA76758 5188 6P pallidum EFA80531 100 15D fasciculatum EGG19875 9998 8D fasciculatum EGG23890 100 12

all four cellular slime mould species However some PPRhomologs could only be found in D discoideum and Dpurpureum indicating a potential conserved function of theproteins in these organisms which is either not requiredor is performed by a different protein in P pallidum andD fasciculatum mitochondria In addition our sequenceanalysis also revealed that some of the cellular slime mouldspossess PPR proteins which are not found in any of theothers (Table 4) These candidates may represent uniquePPR proteins that perform functions only required in these


cellular slime moulds However it is noteworthy to mentionthat one of these proteins XP 003291713 fromD purpureummay have a putative homolog inD discoideum (protein acce-ssion number XP 644522) but no PPR motifs were detectedin the D discoideum protein by TPRpred (data not shown)

4 Conclusions

The presence of PPR proteins in the model eukaryote Ddiscoideum suggests an important role for these proteins inthe regulation of the mitochondrial transcriptome This issupported by the antisense inhibition of one of the PPR-encoding genes ptcB yielding phenotypes characteristic ofmitochondrial dysfunction in the protist While the precisefunction of PPR proteins remains to be elucidated it is clearthat the function of most of these proteins is conservedsupported by the presence of homologs in other cellular slimemoulds The potential functions of these proteins seem todiffer from the function of RNA editing type PPR proteins inN gruberi butmay be analogous to the function of trypanoso-mal PPR proteins in modifying the stability of mitochondrialtranscripts One of the PPR candidates identified PtcE alsocontains a C-terminal methyltransferase domain which hasnot been identified in any PPR protein to date furtherattesting to the significance of studying PPR proteins inthe D discoideum model The potential methyltransferaseactivity and the presence of other domains in some of thePPR proteins therefore suggest some unique functions forPPR proteins in D discoideum mitochondria which havenot been observed for PPR proteins of other organismsbefore Thus the functional study of PPR proteins in Ddiscoideum will provide an elegant system for investigatingthe important role PPR proteins played not only in protozoanmitochondrial gene expression but also more generally innon-plant organisms

Abbreviations

PPR Pentatricopeptide repeatPtcA-L Pentatricopeptide repeat-containing protein A-LPORR Plant organelle RNA recognition

Conflict of Interests

All authors declare that they do not have any conflict of inte-rests with any trademark or softwarementioned in this paper

Acknowledgment

Sam Manna was the recipient of an Australian PostgraduateAward

References

[1] M W Gray B F Lang and G Burger ldquoMitochondria ofprotistsrdquo Annual Review of Genetics vol 38 pp 477ndash524 2004

[2] T E Shutt and G S Shadel ldquoA compendium of human mito-chondrial gene expression machinery with links to diseaserdquo

Environmental and Molecular Mutagenesis vol 51 no 5 pp360ndash379 2010

[3] ID Small andN Peeters ldquoThePPRmotifmdashaTPR-relatedmotifprevalent in plant organellar proteinsrdquo Trends in BiochemicalSciences vol 25 no 2 pp 46ndash47 2000

[4] C Lurin C Andres S Aubourg et al ldquoGenome-wide analysisof Arabidopsis pentatricopeptide repeat proteins reveals theiressential role in organelle biogenesisrdquo Plant Cell vol 16 no 8pp 2089ndash2103 2004

[5] C Schmitz-Linneweber and I Small ldquoPentatricopeptide repeatproteins a socket set for organelle gene expressionrdquo Trends inPlant Science vol 13 no 12 pp 663ndash670 2008

[6] J Pfalz O A Bayraktar J Prikryl and A Barkan ldquoSite-specificbinding of a PPR protein defines and stabilizes 51015840 and 31015840 mRNAtermini in chloroplastsrdquoThe EMBO Journal vol 28 no 14 pp2042ndash2052 2009

[7] E Delannoy W A Stanley C S Bond and I D Small ldquoPen-tatricopeptide repeat (PPR) proteins as sequence-specificityfactors in post-transcriptional processes in organellesrdquo Bio-chemical Society Transactions vol 35 no 6 pp 1643ndash1647 2007

[8] M Rudinger M Polsakiewicz and V Knoop ldquoOrganellarRNA editing and plant-specific extensions of pentatricopeptiderepeat proteins in jungermanniid but not in marchantiid liver-wortsrdquoMolecular Biology and Evolution vol 25 no 7 pp 1405ndash1414 2008

[9] V Salone M Rudinger M Polsakiewicz et al ldquoA hypothesison the identification of the editing enzyme in plant organellesrdquoFEBS Letters vol 581 no 22 pp 4132ndash4138 2007

[10] S Fujii and I Small ldquoThe evolution of RNA editing andpentatricopeptide repeat genesrdquo New Phytologist vol 191 no 1pp 37ndash47 2011

[11] O Rackham and A Filipovska ldquoThe role of mammalian PPRdomain proteins in the regulation of mitochondrial gene expre-ssionrdquo Biochimica et Biophysica Acta vol 1819 no 9-10 pp1008ndash1016 2011

[12] O Rackham T RMercer andA Filipovska ldquoThe humanmito-chondrial transcriptome and the RNA-binding proteins thatregulate its expressionrdquo Wiley Interdisciplinary Reviews RNAvol 3 no 5 pp 675ndash695 2012

[13] M I G L Sanchez T R Mercer S M K Davies et al ldquoRNAprocessing in human mitochondriardquo Cell Cycle vol 10 no 17pp 2904ndash2916 2011

[14] S M K Davies O Rackham A-M J Shearwood et al ldquoPenta-tricopeptide repeat domain protein 3 associates with the mito-chondrial small ribosomal subunit and regulates translationrdquoFEBS Letters vol 583 no 12 pp 1853ndash1858 2009

[15] S M Davies M I L Sanchez R Narsai et al ldquoMRPS27 isa pentatricopeptide repeat domain protein required for thetranslation of mitochondrially encoded proteinsrdquo FEBS Lettersvol 586 no 20 pp 3555ndash3561 2012

[16] I Aphasizheva D Maslov X Wang L Huang and R Apha-sizhev ldquoPentatricopeptide repeat proteins stimulatemrna aden-ylationuridylation to activate mitochondrial translation intrypanosomesrdquoMolecular Cell vol 42 no 1 pp 106ndash117 2011

[17] M K Mingler A M Hingst S L Clement L E Yu L Reifurand D J Koslowsky ldquoIdentification of pentatricopeptide repeatproteins in Trypanosoma bruceirdquo Molecular and BiochemicalParasitology vol 150 no 1 pp 37ndash45 2006

[18] M Pusnik I Small L K Read T Fabbro and A Schnei-der ldquoPentatricopeptide repeat proteins in Trypanosoma bruceifunction in mitochondrial ribosomesrdquo Molecular and CellularBiology vol 27 no 19 pp 6876ndash6888 2007


[19] M Pusnik and A Schneider ldquoA trypanosomal pentatricopep-tide repeat protein stabilizes the mitochondrial mRNAs ofcytochrome oxidase subunits 1 and 2rdquo Eukaryotic Cell vol 11no 1 pp 79ndash87 2012

[20] V Knoop andM Rudinger ldquoDYW-type PPR proteins in a hete-rolobosean protist plant RNA editing factors involved in anancient horizontal gene transferrdquo FEBS Letters vol 584 no 20pp 4287ndash4291 2010

[21] M Rudinger L Fritz-Laylin M Polsakiewicz and V KnoopldquoPlant-type mitochondrial RNA editing in the protistNaegleriagruberirdquo RNA vol 17 no 12 pp 2058ndash2062 2011

[22] S J Annesley and P R Fisher ldquoDictyostelium discoideum-amodel for many reasonsrdquoMolecular and Cellular Biochemistryvol 329 no 1-2 pp 73ndash91 2009

[23] L M Francione S J Annesley S Carilla-Latorre R Escalanteand P R Fisher ldquoThe Dictyostelium model for mitochondrialdiseaserdquo Seminars in Cell and Developmental Biology vol 22no 1 pp 120ndash130 2011

[24] K Angata S Ogawa K Yanagisawa and Y Tanaka ldquoA group-I intron in the mitochondrial large-subunit ribosomal RNA-encoding gene of Dictyostelium discoideum same site localiza-tion in alga and in vitro self-splicingrdquo Gene vol 153 no 1 pp49ndash55 1995

[25] C Barth U Greferath M Kotsifas and P R Fisher ldquoPoly-cistronic transcription and editing of the mitochondrial smallsubunit (SSU) ribosomal RNA in Dictyostelium discoideumrdquoCurrent Genetics vol 36 no 1-2 pp 55ndash61 1999

[26] C Barth U Greferath M Kotsifas et al ldquoTranscript mappingand processing of mitochondrial RNA in Dictyostelium dis-coideumrdquo Current Genetics vol 39 no 5-6 pp 355ndash364 2001

[27] C Barth P Le and P R Fisher ldquoMitochondrial biology anddisease in Dictyosteliumrdquo International Review of Cytology vol263 pp 207ndash252 2007

[28] P Le P R Fisher and C Barth ldquoTranscription of the Dic-tyostelium discoideum mitochondrial genome occurs from asingle initiation siterdquo RNA vol 15 no 12 pp 2321ndash2330 2009

[29] D J Watts and J M Ashworth ldquoGrowth of myxameobae ofthe cellular slime mould Dictyostelium discoideum in axenicculturerdquo Biochemical Journal vol 119 no 2 pp 171ndash174 1970

[30] M Darmon P Brachet and L H P Da Silva ldquoChemotacticsignals induce cell differentiation in Dictyostelium discoideumrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 72 no 8 pp 3163ndash3166 1975

[31] M Sussman ldquoBiochemical and genetic methods in the study ofcellular slime mold developmentrdquoMethods in Cell Biology vol2 no C pp 397ndash410 1966

[32] W Nellen C Silan and R A Firtel ldquoDNA-mediated transfor-mation in Dictyostelium discoideum regulated expression of anactin gene fusionrdquoMolecular and Cellular Biology vol 4 no 12pp 2890ndash2898 1984

[33] Z Wilczynska and P R Fisher ldquoAnalysis of a complex plasmidinsertion in a photoaxis-deficient transformant ofDictyosteliumdiscoideum selected on aMicrococcus luteus lawnrdquo Plasmid vol32 no 2 pp 182ndash194 1994

[34] P R Gilson X-C Yu D Hereld et al ldquoTwo Dictyosteliumorthologs of the prokaryotic cell division protein FtsZ localize tomitochondria and are required for the maintenance of normalmitochondrial morphologyrdquo Eukaryotic Cell vol 2 no 6 pp1315ndash1326 2003

[35] A U Ahmed P L Beech S T Lay P R Gilson and P R FisherldquoImport-associated translational inhibition novel in vivo evi-dence for cotranslational protein import into Dictyostelium

discoideum mitochondriardquo Eukaryotic Cell vol 5 no 8 pp1314ndash1327 2006

[36] P B Bokko L Francione E Bandala-Sanchez et al ldquoDiversecytopathologies in mitochondrial disease are caused by AMP-activated protein kinase signalingrdquoMolecular Biology of the Cellvol 18 no 5 pp 1874ndash1886 2007

[37] M R Karpenahalli A N Lupas and J Soding ldquoTPRpred a toolfor prediction of TPR- PPR- and SEL1-like repeats from proteinsequencesrdquo BMC Bioinformatics vol 8 article 2 2007

[38] T S Kroeger K P Watkins G Friso K J Van Wijk andA Barkan ldquoA plant-specific RNA-binding domain revealedthrough analysis of chloroplast group II intron splicingrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 106 no 11 pp 4537ndash4542 2009

[39] J Jacobs and U Kuck ldquoFunction of chloroplast RNA-bindingproteinsrdquo Cellular and Molecular Life Sciences vol 68 no 5 pp735ndash748 2011

[40] C C Des Francs-Small T KroegerM Zmudjak et al ldquoA PORRdomain protein required for rpl2 and ccmF

119862intron splicing

and for the biogenesis of c-type cytochromes in Arabidopsismitochondriardquo Plant Journal vol 69 no 6 pp 996ndash1005 2012

[41] JM Zapata VMartınez-Garcıa and S Lefebvre ldquoPhylogeny ofthe TRAFMATH domainrdquoAdvances in Experimental Medicineand Biology vol 597 pp 1ndash24 2007

[42] M G Claros and P Vincens ldquoComputational method to pre-dict mitochondrially imported proteins and their targetingsequencesrdquo European Journal of Biochemistry vol 241 no 3 pp779ndash786 1996

[43] W Witke W Nellen and A Noegel ldquoHomologous recombi-nation in the Dictyostelium 120572-actinin gene leads to an alteredmRNA and lack of the proteinrdquo The EMBO Journal vol 6 no13 pp 4143ndash4148 1987

[44] C Barth D J Fraser and P R Fisher ldquoCo-insertional repli-cation is responsible for tandem multimer formation duringplasmid integration into the Dictyostelium genomerdquo Plasmidvol 39 no 2 pp 141ndash153 1998

[45] K Meierhoff S Felder T Nakamura N Bechtold and GSchuster ldquoHCF152 anArabidopsisRNAbinding pentatricopep-tide repeat protein involved in the processing of chloroplastpsbB-psbT-psbH-petB-petD RNAsrdquo Plant Cell vol 15 no 6 pp1480ndash1495 2003

[46] D Sosso S Mbelo V Vernoud et al ldquoPPR2263 a DYW-sub-group Pentatricopeptide repeat protein is required for mito-chondrial nad5 and cob transcript editing mitochondrionbiogenesis andmaize growthrdquo Plant Cell vol 24 no 2 pp 676ndash691 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2013

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2013

ISRN Biotechnology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013


GenomicsInternational Journal of

Volume 2013

Evolutionary BiologyInternational Journal of



Advances in

Virolog y

ISRN Microbiology


Marine BiologyJournal of


BioMed Research International


ISRN Zoology



Signal TransductionJournal of

ISRN Cell Biology



BioinformaticsAdvances in

PeptidesInternational Journal of



Enzyme Research


Biochemistry Research International

ISRN Molecular Biology


Stem CellsInternational



activation [5 7] Although several of these functions areregulated by PPR proteins in plants the most commonfunction for plant PPR proteins seems to be in RNA editing aprocess which is rather common in plant organelles [5 10] Inhumans only seven PPR proteins have been identified Theyhave been shown to regulate themitochondrial transcriptomenot via RNA editing but rather through transcription andtranscript processing RNA stability polyadenylation andtranslation [11ndash15]

While the knowledge of PPR protein structure and fun-ction in non-plant organisms is expanding exponentiallylittle is known about the significance of these proteins in themitochondria of protozoa In the protists PPR proteins havebeen studied mainly in trypanosomatids where more than30 PPR genes have been identified a uniquely high numberfor a non-plant organism [16ndash19] Most of these PPR proteinsplay roles in either the stabilisation or polyadenylation ofkinetoplast transcripts and they often lack additional C-terminal domains [16ndash19] While studies into the heterolo-bosean protistNaegleria gruberi have also identified an unex-pectedly high number of PPR-encoding genes in contrast totrypanosomes a large subset of the gene products belongsto the DYW subclass of the PLS group and has thus beenimplicated in RNA editing [20 21] Despite the identificationof PPR genes in N gruberi none of their gene products havebeen functionally characterised and therefore the questionremains whether transcript stabilisation and editing are themain functions of PPR proteins in protists

Dictyostelium discoideum is a cellular slime mould bel-onging to the Amoebozoa and is a widely accepted and well-established model for studying mitochondrial genetics anddisease [22 23] Transcription of the mitochondrial genomein D discoideum has been studied in detail and some of thecore components mediating the transcription process havebeen identified InD discoideummitochondria transcriptionis initiated at a single site and the transcriptome is subjectedto several post-transcriptional modifications including pro-cessing and intron splicing as well as a single nucleotide RNAediting event that occurs in the transcript of the mitochon-drial rns gene [24ndash28] However very little is known aboutthe proteins that regulate these post-transcriptional eventsand the existence and potential role of PPR proteins in mito-chondrial RNAmetabolism have not been investigated in thisorganism Here we describe the identification of genes of thePPR protein family in D discoideum We found 12 potentialPPR proteins encoded in the D discoideum genome andsome of these proteins show significantly different featurescompared to other known PPR proteins One of the D dis-coideum proteins has been characterised in detail confirmingits mitochondrial localisationWe also demonstrate that anti-sense inhibition of its expression leads to growth defects aphenotype associatedwithmitochondrial dysfunctionWhilethe phenotypic changes resulting from antisense inhibitionof gene expression of one of these PPR proteins confirmthe importance of these proteins in mitochondrial functiontheir specific role in post-transcriptional regulation of the Ddiscoideum mitochondrial transcriptome still remains to bedetermined

2 Materials and Methods

21 Strains and Culture Conditions D discoideum strain AX2and all transformants were grown to a density of 2ndash5 times 106cellsmL in HL-5 medium at 21∘C [29 30] For non-axenicculture AX2 and all derivativeswere grownon SMplateswithKlebsiella aerogenes lawns [31] unless otherwise stated

22 Transformation of D discoideum with Vector DNA Thecalcium phosphate precipitation method was used to trans-formD discoideumwith vector DNA as described previously[32] using 20120583g of vector DNA Transformants were isolatedonMicrococcus luteus lawns on SM plates supplemented with20120583gmL G-418 [33]

23 Fluorescence Microscopy To determine the subcellularlocalisation of PtcB D discoideum transformants expressinga PtcBGFP fusion protein were analysed via fluorescencemicroscopy as described previously [34 35] Aliquots ofthe axenically grown transformant culture (sim3mL) weretransferred into a 6-well plate (BD Biosciences) containingcoverslips and the cells were allowed to settle The mediumwas removed and themitochondria were stainedwith 100 nMMitoTracker (Life Technologies) in Lo-Flo HL-5 medium for1 hour Unbound MitoTracker was removed by washing thecells four times with Lo-Flo HL-5 and twice with phosphatebuffer The cells were subsequently fixed by placing thecoverslips for 15 minutes upside down onto a 1 agarosegel in phosphate buffer containing 37 paraformaldehydeafter which the cells were washed four times with phosphatebuffered saline (PBS) Coverslips were rinsed with Milli-QsdH2O and mounted for microscopy with 90 glycerol in

PBS

24 Analysis of Growth Rates on Bacterial Lawns Growthof D discoideum cells was analysed by measuring plaqueexpansion rates on bacterial lawns as described previously[36] Briefly D discoideum cells of interest were collectedfrom the leading edge of a previously grown plaque onK aerogenes lawns The cells were then used to inoculatenormal agar plates with pregrown Escherichia coli B2 lawnsThe diameter of the plaques was measured every 8 or 16hours for 7 days to calculate the mean plaque expansion rate(mmhour) as an estimate of growth

25 Quantitative PCR The number of vector copies of theptcB antisense construct in each transformant was deter-mined using qPCR The qPCR reactions were performedusing SsoAdvanced SYBR Green Supermix (Bio-Rad) TotalgDNA extracted from each antisense transformant and fromwild type cells was used as template along with primersspecific to the cloned portion of the ptcB gene Cyclingconditions were as follows initial denaturation at 95∘C for10 minutes and then 40 cycles of denaturation at 95∘C for15 seconds followed by annealing and primer extension at60∘C for 1 minute All transcript levels were normalised tothe single copy number 120573-tubulin (tubB) gene






Number ofPPR motifs



















Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

6262627363536227

115115115131108596287

169169169185166656347

219219219239223716405

lowast lowast

lowast








(a) (b) (c)




035

04

045

05

055

06

065

07

0 50 100 150 200 250 300 350

Plaq

ue ex

pans

ion

rate

(mm

hr)


R2= 07301






D discoideumprotein

D purpureumhomolog

P pallidumhomolog



PtcB XP 003288427(529712)

EFA79424(16227)

EGG14329(321494)

PtcC XP 003290170(481666)

EFA76720(374555)

EGG22645(393621)


PtcE XP 003288663(675785)

EFA82229(463609)

EGG13534(175246)

PtcF XP 003294037(499648)

EFA79525(275503)

EGG15096(2949)

PtcG XP 003284179(619751)

EFA75260(28237)

EGG14213(495648)










PPRprobability ()

Number ofPPR motifs




PPRprobability ()

Number ofPPR motifs





4 Conclusions


Abbreviations




Acknowledgment


References

























































Volume 2013


ISRN Biotechnology




Volume 2013




Advances in

Virolog y

ISRN Microbiology






ISRN Zoology




ISRN Cell Biology







Enzyme Research












Number ofPPR motifs



















Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

6262627363536227

115115115131108596287

169169169185166656347

219219219239223716405

lowast lowast

lowast








(a) (b) (c)




035

04

045

05

055

06

065

07

0 50 100 150 200 250 300 350

Plaq

ue ex

pans

ion

rate

(mm

hr)


R2= 07301






D discoideumprotein

D purpureumhomolog

P pallidumhomolog



PtcB XP 003288427(529712)

EFA79424(16227)

EGG14329(321494)

PtcC XP 003290170(481666)

EFA76720(374555)

EGG22645(393621)


PtcE XP 003288663(675785)

EFA82229(463609)

EGG13534(175246)

PtcF XP 003294037(499648)

EFA79525(275503)

EGG15096(2949)

PtcG XP 003284179(619751)

EFA75260(28237)

EGG14213(495648)










PPRprobability ()

Number ofPPR motifs




PPRprobability ()

Number ofPPR motifs





4 Conclusions


Abbreviations




Acknowledgment


References

























































Volume 2013


ISRN Biotechnology




Volume 2013




Advances in

Virolog y

ISRN Microbiology






ISRN Zoology




ISRN Cell Biology







Enzyme Research












Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

Hs UBHMm UBH

Bt UBHGg UBHSc UBH

Dd PtcKZm WTF1

6262627363536227

115115115131108596287

169169169185166656347

219219219239223716405

lowast lowast

lowast








(a) (b) (c)




035

04

045

05

055

06

065

07

0 50 100 150 200 250 300 350

Plaq

ue ex

pans

ion

rate

(mm

hr)


R2= 07301






D discoideumprotein

D purpureumhomolog

P pallidumhomolog



PtcB XP 003288427(529712)

EFA79424(16227)

EGG14329(321494)

PtcC XP 003290170(481666)

EFA76720(374555)

EGG22645(393621)


PtcE XP 003288663(675785)

EFA82229(463609)

EGG13534(175246)

PtcF XP 003294037(499648)

EFA79525(275503)

EGG15096(2949)

PtcG XP 003284179(619751)

EFA75260(28237)

EGG14213(495648)










PPRprobability ()

Number ofPPR motifs




PPRprobability ()

Number ofPPR motifs





4 Conclusions


Abbreviations




Acknowledgment


References

























































Volume 2013


ISRN Biotechnology




Volume 2013




Advances in

Virolog y

ISRN Microbiology






ISRN Zoology




ISRN Cell Biology







Enzyme Research








(a) (b) (c)




035

04

045

05

055

06

065

07

0 50 100 150 200 250 300 350

Plaq

ue ex

pans

ion

rate

(mm

hr)


R2= 07301






D discoideumprotein

D purpureumhomolog

P pallidumhomolog



PtcB XP 003288427(529712)

EFA79424(16227)

EGG14329(321494)

PtcC XP 003290170(481666)

EFA76720(374555)

EGG22645(393621)


PtcE XP 003288663(675785)

EFA82229(463609)

EGG13534(175246)

PtcF XP 003294037(499648)

EFA79525(275503)

EGG15096(2949)

PtcG XP 003284179(619751)

EFA75260(28237)

EGG14213(495648)










PPRprobability ()

Number ofPPR motifs




PPRprobability ()

Number ofPPR motifs





4 Conclusions


Abbreviations




Acknowledgment


References

























































Volume 2013


ISRN Biotechnology




Volume 2013




Advances in

Virolog y

ISRN Microbiology






ISRN Zoology




ISRN Cell Biology







Enzyme Research









D discoideumprotein

D purpureumhomolog

P pallidumhomolog



PtcB XP 003288427(529712)

EFA79424(16227)

EGG14329(321494)

PtcC XP 003290170(481666)

EFA76720(374555)

EGG22645(393621)


PtcE XP 003288663(675785)

EFA82229(463609)

EGG13534(175246)

PtcF XP 003294037(499648)

EFA79525(275503)

EGG15096(2949)

PtcG XP 003284179(619751)

EFA75260(28237)

EGG14213(495648)










PPRprobability ()

Number ofPPR motifs




PPRprobability ()

Number ofPPR motifs





4 Conclusions


Abbreviations




Acknowledgment


References

























































Volume 2013


ISRN Biotechnology




Volume 2013




Advances in

Virolog y

ISRN Microbiology






ISRN Zoology




ISRN Cell Biology







Enzyme Research









4 Conclusions


Abbreviations




Acknowledgment


References

























































Volume 2013


ISRN Biotechnology




Volume 2013




Advances in

Virolog y

ISRN Microbiology






ISRN Zoology




ISRN Cell Biology







Enzyme Research












































Volume 2013


ISRN Biotechnology




Volume 2013




Advances in

Virolog y

ISRN Microbiology






ISRN Zoology




ISRN Cell Biology







Enzyme Research












Volume 2013


ISRN Biotechnology




Volume 2013




Advances in

Virolog y

ISRN Microbiology






ISRN Zoology




ISRN Cell Biology







Enzyme Research







Documents

Identification of Pentatricopeptide Repeat Proteins in the ... · Identification of Pentatricopeptide Repeat Proteins in the Model Organism Dictyostelium discoideum ... (bp) Length