Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-0

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>n mRNA contains a 92 bp extension at the 5' side depicted in white. Primers used for RT-PCR are indicated by arrows. Primers 246 and 247 are complementary to the sequences around the two start ATG codons, 248 binds at the sequence around the TAA stop codon. . RT-PCR products with mRNA from liver, kidney and Caco-2 cells after 30 amplification cycles. Primers 246 and 248 yield a 488 bp PCR product only on those Par14 mRNAs with 5' extension. Primers 247 and 248 give rise to a 385 bp DNA fragment with all Par14 mRNAs. All longer RT-PCR products were eluted from the gel and sequenced. . Two start ATG codons are indicated in bold capital letters. The sequence of Parvulin common to both originally described cDNAs by Uchida . [GenBank:] [3] and Rulten . [GenBank:] [9] begins at the caa codon depicted in bold. The peptide sequence used for antibody production is shaded in grey. Two SNPs are shown leading to amino acid substitutions Q16R and R18S

Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-4

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>sequence two additional in-frame ATG codons (highlighted in blue). The only other 5' ATG codon is present in the sequence; it is out of frame and followed by a TAA stop codon (both underlined). All in-frame stop codons are marked in red. Only the human sequence displays an extended open reading frame, both as QR and RS isoform (SNPs highlighted in grey)

Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation-2

<p><b>Copyright information:</b></p><p>Taken from "Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation"</p><p>BMC Molecular Biology 2006;7():9-9.</p><p>Published online 7 Mar 2006</p><p>PMCID:PMC1420321.</p><p></p>. Reaction products were separated by 17.5% SDS-PAGE following autoradiography. . Equal amounts of 35S-methionine labeled Par17-QR, Par17-RS and Par14 were incubated with increasing concentrations of proteinaseK (PK) for 30 min at 10°C. Enzyme concentrations are given in μg/ml. Reactions were stopped by an excess of PMSF, separated by SDS-PAGE and subjected to autoradiography. . Western blot of HepG2 (lane 1 and 3) and HeLa (lane 2 and 4) cell lysates. Proteins were separated by SDS-PAGE with MES as running buffer and SeeBlue2 as protein standard, transferred to nitrocellulose membranes. Blots were incubated with pre-immune (lane 1 and 2) or anti-Par17 serum (lane 3 and 4; both at 500-fold dilution). . Coding sequences for Par17-RS and -QR were subcloned in the pET-28 vector with N-terminal His6 tag and expressed in . Lysates were subjected to reducing SDS-PAGE in MES buffer and SeeBlue2 as protein standard, transferred to nitrocellulose membranes and incubated with the anti-Par17 antibody. -, before IPTG induction; RS, Par17-RS lysates; QR, Par17-QR lysates. Coomassie stained gel of lysates to show equal loading. Par17 fusions with His6 tag and thrombin cleavage sites show apparent molecular weights of about 22 KDa in SDS-PAGE. The induction band in E. coli lysates and the corresponding band recognized by Ab-EXT are labeled with arrows. The lower migrating band may be caused by proteolytic degradation. . Par14 coding sequence was expressed as GFP fusion in HeLa cells (lane 3). Lane 1 and 2 are HeLa cell lysates not transfected with this construct. Lysates were subjected to reducing SDS-PAGE in Tris-glycine buffer with MagicMark as protein standard, transferred to nitrocellulose membranes and incubated with anti-Par17 and anti-GFP antibodies. Coomassie stained gel of lysates to show equal loading

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae-5

<p><b>Copyright information:</b></p><p>Taken from "The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae"</p><p>http://www.biomedcentral.com/1741-7007/5/37</p><p>BMC Biology 2007;5():37-37.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2031878.</p><p></p>s. The resulting DNA sequences were aligned with respect to the Par17 and Par14 ATG codons; the respective sequence was obtained from Ensembl and aligned accordingly. Translated protein sequences in-frame with the respective ATG codons are given below the DNA sequences with stop codons written in red. Within the genomic sequences of and (obtained from Ensembl), the Par17 ATG codons are immediately preceded by an in-frame stop codon. The Q16/R18 to R16/S18 coupled SNPs that were described for the human sequence are highlighted in grey; comparison with the other primate sequences reveals the QR form to be the ancestral one. Phylogenetic clades used in scheme B are indicated by boxes here and the respective abbreviations from B. B. Phylogenetic scheme of primate relationships and Par17 sequence features. The Par17 elongation is preformed in sequence in all anthropoid genomic DNAs tested. Additional ATG codons at the putative starting position of Par17 exist in all tested species, but are out-of-frame relative to the Par14 coding sequence in New World and Old World monkeys. The gap of five nucleotides in New World monkeys shrinks to a gap of four nucleotides in Old World monkeys, still preventing a continuous ORF with Par14. The upstream ATG codons are in-frame with the Par14 sequence in the clade of apes, only. Within the gibbon sequence however, an in-frame stop codon prevents expression of an extended protein. This stop codon has been sequenced in three independent clones. Thus, only Hominidae (and ) possess an extended ORF that can lead to the expression of Par17. (The length of the branches in this scheme does not reflect any phylogenetic distances.) C. Exon 3 sequences were obtained as in A. Exon 3 sequences are highly conserved with only two amino acid substitutions between human and cow or human and mouse, respectively. Amino acid exchanges relative to the human sequence are in white on black. Phylogenetic groupings are indicated by boxes as in A

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae-3

<p><b>Copyright information:</b></p><p>Taken from "The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae"</p><p>http://www.biomedcentral.com/1741-7007/5/37</p><p>BMC Biology 2007;5():37-37.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2031878.</p><p></p>ne labeled Par17 and Par14 lysates for 15 min at 25°C; Porin and GFP lysates were used as controls. Mitochondria were separated from soluble protein by the addition 500 mM sucrose buffer, subsequent centrifugation and additional washing in a 250 mM sucrose containing SEM buffer to ensure efficient separation of mitochondria from lysate ingredients. Mitochondria were then suspended in buffer containing increasing salt concentrations (0, 80 and 600 mM NaCl) and re-isolated by centrifugation. The samples within each panel including the respective reference sample (L, 20% for Porin and GFP, 50% for Par14 and Par17) were separated on the same gel to allow direct comparison (M +, mitochondria added). The amount of associated radioactively labeled protein was analyzed by SDS-PAGE and autoradiography. Mitochondrial association of Par14 and Par17 as displayed as bars on the diagram represent data normalized to the highest value including error bars (standard deviation for n = 3). B. Par17, but not Par14, is imported into isolated human, rat and yeast mitochondria. Left. Par17-QR and Par14 S-methionine labeled lysates were incubated with different amounts of PK showing complete degradation of soluble parvulin proteins at a PK concentration of 50 μg/ml; at least 100 μg/ml PK were used for all following experiments. Right. Radiolabeled Par17-QR was incubated with mitochondria from human Jurkat cells, rat liver or from yeast either with or without subsequent proteinase K (PK) treatment (at least 100 μg/ml for 10 min). Mitochondria were then re-isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. Par17 reached a PK protected compartment irrespective of the source of mitochondria indicating mitochondrial import. Par14, also present in this reaction, could be re-isolated together with mitochondria during centrifugation, but did not reach a protease-protected compartment. C. Time dependence of Par17 mitochondrial import. Mitochondria from yeast and human Jurkat cells were incubated with Par17-QR lysates for the times indicated followed by PK treatment. To assess relative import efficiencies, lysates of Su9-DHFR (amino acids 1–69 from subunit 9 of ATP synthetase fused to mouse DHFR [28]) were imported into the same mitochondrial preparations for equal time intervals at identical reaction conditions. Import reactions of Par17-QR and mature Su9-DHFR were analyzed by autoradiography and densitometric quantifications. Values were expressed as percent of added protein with correction for the different numbers of methionine residues in the precursor and processed proteins of Su9-DHFR. D. Par17 import depends on the mitochondrial membrane potential. Import reactions were performed as in B (right panel). Mitochondria were treated with the uncoupling reagent valinomycin to dissipate the mitochondrial membrane potential either before or after import reaction. Par17 was only imported into mitochondria with intact membrane potential (without valinomycin; or when the uncoupling agent was added after import). E. Par17 reaches the inner membrane of rat mitochondria. Porin and Par17-QR were imported into mitochondria isolated from rat liver. The outer mitochondrial membrane was removed by hypo-osmotic swelling as indicated by the disappearance of the porin signal. The Par17 band however only disappeared upon complete lysis of mitochondria by digitonin indicating that Par17 was imported at least to the inner mitochondrial membrane. F. Par17 is imported into the mitochondrial matrix. Import reactions with yeast mitochondria were performed with increasing amounts of digitonin leading to stepwise lysis. The amounts of Par17-QR, the inner membrane protein Tim23 and the matrix protein Mge1 were monitored in parallel samples using autoradiography for Par17 and Western blotting for Tim23 and Mge1. Tim23 is already degraded at a concentration of 0.1% digitonin, while Par17-QR and the matrix protein Mge1 resist PK. Degradation of these two proteins is only observed upon addition of 0.2% digitonin indicating that Par17 is transported to the mitochondrial matrix. Gels (upper part) and densitometric analysis (lower part) are shown

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae-4

<p><b>Copyright information:</b></p><p>Taken from "The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae"</p><p>http://www.biomedcentral.com/1741-7007/5/37</p><p>BMC Biology 2007;5():37-37.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2031878.</p><p></p>ncubation of dsDNA with protein lysates, bound protein was eluted with increasing KCl concentrations. 20% of lysate (Lys) and KCl fractions were analyzed by SDS-PAGE and autoradiography. Asterisk (*), unspecific band caused by free label. Prominent signals for both, Par17 and Par14, are seen at 100 and 200 mM KCl. Porin was used as negative control and was washed off at 0 and 50 mM KCl. B. Quantitative analysis. Par17-QR, -RS and Par14 fused to EGFP as well as EGFP without fusions were subjected to the DNA binding assay. Blots were quantitatively analyzed by densitometry. Data from each experiment were normalized to their sum. Standard deviations are given from duplicate experiments. Par17 and Par14 proteins were eluted from dsDNA cellulose at 100, 200 and to a lesser extent at 500 mM KCl pointing to similar DNA-binding of the two parvulin proteins at physiological salt concentrations. EGFP without fusions was completely eluted from DNA cellulose at 50 mM KCl

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae-0

<p><b>Copyright information:</b></p><p>Taken from "The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae"</p><p>http://www.biomedcentral.com/1741-7007/5/37</p><p>BMC Biology 2007;5():37-37.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2031878.</p><p></p>s. The resulting DNA sequences were aligned with respect to the Par17 and Par14 ATG codons; the respective sequence was obtained from Ensembl and aligned accordingly. Translated protein sequences in-frame with the respective ATG codons are given below the DNA sequences with stop codons written in red. Within the genomic sequences of and (obtained from Ensembl), the Par17 ATG codons are immediately preceded by an in-frame stop codon. The Q16/R18 to R16/S18 coupled SNPs that were described for the human sequence are highlighted in grey; comparison with the other primate sequences reveals the QR form to be the ancestral one. Phylogenetic clades used in scheme B are indicated by boxes here and the respective abbreviations from B. B. Phylogenetic scheme of primate relationships and Par17 sequence features. The Par17 elongation is preformed in sequence in all anthropoid genomic DNAs tested. Additional ATG codons at the putative starting position of Par17 exist in all tested species, but are out-of-frame relative to the Par14 coding sequence in New World and Old World monkeys. The gap of five nucleotides in New World monkeys shrinks to a gap of four nucleotides in Old World monkeys, still preventing a continuous ORF with Par14. The upstream ATG codons are in-frame with the Par14 sequence in the clade of apes, only. Within the gibbon sequence however, an in-frame stop codon prevents expression of an extended protein. This stop codon has been sequenced in three independent clones. Thus, only Hominidae (and ) possess an extended ORF that can lead to the expression of Par17. (The length of the branches in this scheme does not reflect any phylogenetic distances.) C. Exon 3 sequences were obtained as in A. Exon 3 sequences are highly conserved with only two amino acid substitutions between human and cow or human and mouse, respectively. Amino acid exchanges relative to the human sequence are in white on black. Phylogenetic groupings are indicated by boxes as in A