ITRM20100696A1 - PEPTIDIC MOLECULES FOR THE TREATMENT OF MITOCONDRIAL DISEASES - Google Patents
PEPTIDIC MOLECULES FOR THE TREATMENT OF MITOCONDRIAL DISEASES Download PDFInfo
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- ITRM20100696A1 ITRM20100696A1 IT000696A ITRM20100696A ITRM20100696A1 IT RM20100696 A1 ITRM20100696 A1 IT RM20100696A1 IT 000696 A IT000696 A IT 000696A IT RM20100696 A ITRM20100696 A IT RM20100696A IT RM20100696 A1 ITRM20100696 A1 IT RM20100696A1
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- 229960005542 ethidium bromide Drugs 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 102000043538 human LARS2 Human genes 0.000 description 1
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- 238000000338 in vitro Methods 0.000 description 1
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- 208000006443 lactic acidosis Diseases 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 108010057952 lysyl-phenylalanyl-lysine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 230000025608 mitochondrion localization Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000002232 neuromuscular Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
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- 238000002741 site-directed mutagenesis Methods 0.000 description 1
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Classifications
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/53—Ligases (6)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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Description
Descrizione Description
“Molecole peptidiche per il trattamento di patologie mitocondriali†⠀ œPeptide molecules for the treatment of mitochondrial diseasesâ €
Le malattie mitocondriali umane comprendono un ampio gruppo di patologie gravi (soprattutto neuromuscolari) con meccanismi molecolari poco noti, per le quali non esiste terapia. La frequenza à ̈ senz’altro sottostimata, vista la difficoltà della diagnosi, ma à ̈ dell’ordine di almeno 1:5000, per quanto riguarda quelle dovute a mutazioni nei geni mitocondriali per tRNA, che d’altra parte sono le più frequenti. Sostituzioni di basi nei geni mitocondriali per i tRNA producono nell’uomo gravissime malattie neurodegenerative, per le quali non esiste ad oggi alcuna cura possibile. Human mitochondrial diseases comprise a large group of severe (mainly neuromuscular) diseases with little known molecular mechanisms, for which there is no therapy. The frequency is certainly underestimated, given the difficulty of the diagnosis, but it is of the order of at least 1: 5000, as regards those due to mutations in the mitochondrial genes for tRNA, which on the other hand are the more frequent. Base substitutions in mitochondrial genes for tRNAs produce very serious neurodegenerative diseases in humans, for which there is currently no possible cure.
Vari studi dimostrano l’utilità del modello lievito in questo settore. Infatti, sostituzioni equivalenti a quelle ritenute patologiche sono state introdotte nel DNA mitocondriale di lievito S. cerevisiae mediante trasformazione biolistica e sono stati valutati i difetti dovuti alle mutazioni e la possibilità di correggerli. Various studies demonstrate the usefulness of the yeast model in this sector. In fact, substitutions equivalent to those considered pathological were introduced into the mitochondrial DNA of S. cerevisiae yeast by means of biolistic transformation and the defects due to mutations were evaluated and the possibility of correcting them.
Gli autori della presente invenzione hanno già dimostrato che le aminoacil-tRNA sintetasi (aa-RS, enzimi che catalizzano il legame dell’aminoacido al corrispondente tRNA) umane e/o di lievito sopprimono i difetti respiratori provocati dalle sostituzioni di basi nei tRNA mitocondriali (E.Zennaro, S.Francisci, A.Ragnini, L.Frontali and M.Bolotin-Fukuhara. 1989. A point mutation in a mitochondrial tRNA gene abolishes its 3' end processing NAR, 17,5751-5764; M. Feuermann, S. Francisci, T.Rinaldi, C.De Luca, H.Rohou, L. Frontali M.Bolotin-Fukuhara 2003 The yeast counterparts of human †̃MELAS’mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu EMBO Rep,4, 53-58; C. De Luca ,C. Besagni, L. Frontali , M. Bolotin-Fukuhara, S. Francisci 2006, Mutations in yeast mt tRNAs: Specific and general suppressionby nuclear encoded tRNA interactors,Gene 377 , 169–176; C. De Luca, YF Zhou, A. Montanari, V. Morea, R. Oliva, C. Besagni, M. Bolotin-Fukuhara, L. Frontali, S. Francisci 2009 Can yeast be used to study mitochondrial diseases? Biolistic tRNA mutants for the analysis of mechanisms and suppressors. Mitochondrion, 9 408–417; A. Montanari , C. De Luca , L. Frontali, S. Francisci 2010 Aminoacyl-tRNA synthetases are multivalent suppressors of defects due to human equivalent mutations in yeast mt tRNA genes Biochimica et Biophysica Acta 1803, 1050–1057). The authors of the present invention have already demonstrated that human and / or yeast aminoacyl-tRNA synthetases (aa-RS, enzymes that catalyze the binding of the amino acid to the corresponding tRNA) suppress respiratory defects caused by base substitutions in mitochondrial tRNAs. (E.Zennaro, S.Francisci, A.Ragnini, L.Frontali and M.Bolotin-Fukuhara. 1989. A point mutation in a mitochondrial tRNA gene abolishes its 3 'end processing NAR, 17,5751-5764; M. Feuermann , S. Francisci, T. Rinaldi, C. De Luca, H. Rohou, L. Frontali M. Bolotin-Fukuhara 2003 The yeast counterparts of human â € ̃MELASâ € ™ mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu EMBO Rep, 4, 53-58; C. De Luca, C. Besagni, L. Frontali, M. Bolotin-Fukuhara, S. Francisci 2006, Mutations in yeast mt tRNAs: Specific and general suppression by nuclear encoded tRNA interactors, Gene 377, 169â € “176; C. De Luca, YF Zhou, A. Montanari, V. Morea, R. Oliva, C. Besagni, M. Bolotin-Fukuhara, L. Frontali, S. Francisci 2009 Can yeast be used to study mitochondrial diseases? Biolistic tRNA mutants for the analysis of mechanisms and suppressors. Mitochondrion, 9 408â € “417; A. Montanari, C. De Luca, L. Frontali, S. Francisci 2010 Aminoacyl-tRNA synthetases are multivalent suppressors of defects due to human equivalent mutations in yeast mt tRNA genes Biochimica et Biophysica Acta 1803, 1050â € “1057).
Le aminoacil-tRNA sintetasi (aa-RS) sono enzimi largamente studiati, su cui si basa la fedeltà della sintesi proteica. Di interesse particolare à ̈ la leucil-RS mitocondriale di lievito, o Nam2p, della quale à ̈ stata dimostrata una funzione anche nella maturazione di un introne mitocondriale. Ciò indica una capacità di interazione con specifiche sequenze/strutture di RNA, conservata anche nel gene ortologo umano LARS2, anche se i geni mitocondriali, nell’uomo, non presentano introni. I due enzimi conservano domini altamente omologhi. (Houman F, Rho SB, Zhang J, Shen X, Wang CC, Schimmel P, Martinis SA. 2000 A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria Proc Natl Acad Sci U S A.97, 13743-13748). Aminoacyl-tRNA synthetases (aa-RS) are widely studied enzymes, on which the fidelity of protein synthesis is based. Of particular interest is the yeast mitochondrial leucil-RS, or Nam2p, which has also been shown to function in the maturation of a mitochondrial intron. This indicates a capacity for interaction with specific RNA sequences / structures, also conserved in the human orthologous LARS2 gene, even if the mitochondrial genes in humans do not have introns. The two enzymes retain highly homologous domains. (Houman F, Rho SB, Zhang J, Shen X, Wang CC, Schimmel P, Martinis SA. 2000 A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria Proc Natl Acad Sci U S A.97, 13743-13748 ).
Gli autori hanno recentemente dimostrato che la sovraespressione di NAM2 o di LARS2 in cellule mutate, umane o di lievito, allevia il difetto respiratorio. L’effetto soppressivo si verifica non solo nei confronti di mutazioni del tRNAleu ma anche del tRNAval e del tRNAile: infatti i mutanti del tRNAval e del tRNAile (rispettivamente equivalenti alle sostituzioni omoplasmiche mitocondriali nell’uomo, m. 1624 C→T e m. 4291 T→C ) sono soppressi oltre che dalla specifica sintetasi anche da NAM2p. Ciò potrebbe indicare che sequenze conservate tra le due sintetasi siano in grado di stabilizzare i due tRNA mutati, anche indipendentemente dall’attività catalitica. (A. Montanari , C. De Luca , L. Frontali, S. Francisci 2010 Aminoacyl-tRNA synthetases are multivalent suppressors of defects due to human equivalent mutations in yeast mt tRNA genes Biochimica et Biophysica Acta 1803 1050–1057). Ciò conferma precedenti risultati che avevano dimostrato che la soppressione non à ̈ connessa all’attività catalitica delle aminoacil-tRNA sintetasi. I risultati ottenuti sovraesprimendo versioni di Nam2p mutate nel sito catalitico (C. De Luca ,YF Zhou , A. Montanari, V. Morea , R. Oliva, C. Besagni, M. Bolotin-Fukuhara, L. Frontali, S. Francisci 2009 Can yeast be used to study mitochondrial diseases? Biolistic tRNA mutants for the analysis of mechanisms and suppressors Mitochondrion 9 408–417) fanno ritenere che l’effetto soppressivo non sia legato all’attività catalitica, ma piuttosto alla capacità dell’enzima di riconoscere e interagire con il tRNA mutato stabilizzandone la struttura alterata. L’enzima svolgerebbe quindi una funzione tipo chaperonina, che potrebbe esplicarsi anche su tRNA non specifici per la sintetasi in questione. La soppressione può essere ottenuta anche su cibridi (cellule umane immortalizzate in coltura in cui sono stati introdotti i mitocondri del paziente) o cellule di pazienti in coltura. The authors recently demonstrated that overexpression of NAM2 or LARS2 in mutated human or yeast cells alleviates respiratory defect. The suppressive effect occurs not only against mutations of tRNAleu but also of tRNAval and tRNAile: in fact the mutants of tRNAval and tRNAile (respectively equivalent to mitochondrial homoplasmic substitutions in man, m. 1624 Câ † 'T and m. 4291 Tâ † 'C) are suppressed not only by the specific synthetase but also by NAM2p. This could indicate that conserved sequences between the two synthetases are able to stabilize the two mutated tRNAs, even independently of the catalytic activity. (A. Montanari, C. De Luca, L. Frontali, S. Francisci 2010 Aminoacyl-tRNA synthetases are multivalent suppressors of defects due to human equivalent mutations in yeast mt tRNA genes Biochimica et Biophysica Acta 1803 1050â € “1057). This confirms previous results which showed that the suppression is not related to the catalytic activity of aminoacyl-tRNA synthetases. The results obtained by overexpressing versions of Nam2p mutated in the catalytic site (C. De Luca, YF Zhou, A. Montanari, V. Morea, R. Oliva, C. Besagni, M. Bolotin-Fukuhara, L. Frontali, S. Francisci 2009 Can yeast be used to study mitochondrial diseases? Biolistic tRNA mutants for the analysis of mechanisms and suppressors Mitochondrion 9 408â € “417) suggest that the suppressive effect is not linked to the catalytic activity, but rather to the capacity of enzyme to recognize and interact with mutated tRNA stabilizing its altered structure. The enzyme would therefore perform a chaperonin-like function, which could also be expressed on non-specific tRNAs for the synthetase in question. Suppression can also be achieved on cybrids (immortalized human cells in culture into which the patient's mitochondria have been introduced) or patient cells in culture.
Gli autori ora hanno sorprendentemente e vantaggiosamente trovato che la soppressione può essere ottenuta anche con domini isolati della leucil-tRNA sintetasi, di dimensioni ridotte. La struttura e le attività catalitiche dei diversi domini isolati sono state studiate in dettaglio ( Jennifer L. Hsu, Seung Bae Rho, Kevin M. Vannella, and Susan A. Martinis 2006 Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles J. Biol. Chem. 281: 23075-23082), senza menzione all’attività di soppressione di essi. Gli autori hanno subclonato in appropriati vettori di lievito diversi domini isolati o versioni tronche e hanno studiato l’effetto †curativo†nei confronti di mutanti “patologici†di lievito. Tra i domini saggiati uno, la regione carbossiterminale, ha dimostrato un importante effetto soppressivo. Questo dominio à ̈ composto da circa 60 aa ripiegati in un dominio compatto formato da 4 foglietti beta e due alfa eliche, come mostrato in Tabella 1. The authors have now surprisingly and advantageously found that suppression can also be achieved with isolated domains of leucyl-tRNA synthase, of reduced size. The structure and catalytic activities of the different isolated domains have been studied in detail (Jennifer L. Hsu, Seung Bae Rho, Kevin M. Vannella, and Susan A. Martinis 2006 Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles J. Biol. Chem. 281: 23075-23082), without mentioning their suppression activity. The authors subcloned several isolated domains or truncated versions into appropriate yeast vectors and studied the â € œhealingâ € effect against â € œpathologicalâ € mutants of yeast. Among the domains tested, one, the carboxyterminal region, demonstrated an important suppressive effect. This domain is composed of about 60 aa folded into a compact domain consisting of 4 beta sheets and two alpha helices, as shown in Table 1.
Tab 1. Allineamento multiplo di domini C-terminali di 7 diverse Leucil-tRNA sintetasi batteriche con quella mitocondriale umana (lrshum) (da M. Tukalo, A. Yaremchuk, R. Fukunaga, S. Yokoyama, S. Cusack (2005) The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer–editing conformation Nature Structural & Molecular Biology , 12, 923-930) Tab 1. Multiple alignment of C-terminal domains of 7 different bacterial Leucyl-tRNA synthetases with the human mitochondrial one (lrshum) (from M. Tukalo, A. Yaremchuk, R. Fukunaga, S. Yokoyama, S. Cusack (2005) crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer⠀ "editing conformation Nature Structural & Molecular Biology, 12, 923-930)
Il dominio C terminale à ̈ di 66 aa nella proteina di lievito e di 67 aa in quella umana, e può essere ulteriormente ridotto mantenendo le capacità soppressive; infatti, solo parte del peptide C-terminale contatta direttamente il tRNA. Si conferma altresì che la parte catalitica dell’enzima e la parte di “editing†(controllo e correzione dei tRNA non correttamente acilati) non sono implicate nella soppressione del difetto. L’identificazione di peptidi che curano il difetto strutturale del tRNA à ̈ un’indicazione della loro attività terapeutica per il trattamento delle malattie mitocondriali, dovute a mutazioni nei geni per i tRNA mitocondriali. Finora infatti gli effetti in lievito di mutazioni equivalenti alle umane sono risultati largamente sovrapponibili con quelli ottenuti in colture di cellule provenienti da pazienti. L’invenzione identifica pertanto un gruppo di peptidi capaci di alleviare i difetti respiratori mitocondriali e consente di progettare e validare farmaci per la terapia e prevenzione di queste malattie. The C-terminal domain is 66 aa in yeast protein and 67 aa in human, and can be further reduced while maintaining suppressive capabilities; in fact, only part of the C-terminal peptide directly contacts the tRNA. It is also confirmed that the catalytic part of the enzyme and the "editing" part (control and correction of incorrectly acylated tRNAs) are not involved in the suppression of the defect. The identification of peptides that cure the structural defect of the tRNA is an indication of their therapeutic activity for the treatment of mitochondrial diseases, due to mutations in the mitochondrial tRNA genes. In fact, until now the effects of mutations equivalent to humans in yeast have been found to be largely overlapping with those obtained in cell cultures from patients. The invention therefore identifies a group of peptides capable of alleviating mitochondrial respiratory defects and allows the design and validation of drugs for the therapy and prevention of these diseases.
Forma pertanto oggetto della presente invenzione una molecola peptidica in grado di sopprimere il difetto respiratorio di cellule di lievito mutate in un gene mitocondriale codificante per un tRNA caratterizzata dal fatto di avere una sequenza amminoacidica compresa nel dominio C-terminale di una Leucil-tRNA sintetasi mitocondriale. Preferibilmente la Leucil-tRNA sintetasi mitocondriale à ̈ di origine umana o di lievito. Therefore, the subject of the present invention is a peptide molecule capable of suppressing the respiratory defect of yeast cells mutated in a mitochondrial gene coding for a tRNA characterized by the fact of having an amino acid sequence included in the C-terminal domain of a mitochondrial Leucyl-tRNA synthetase . Preferably the mitochondrial Leucyl-tRNA synthase is of human or yeast origin.
In un aspetto preferito la molecola peptidica à ̈ compresa nella sequenze SEQ ID N. 2 o SEQ ID N.4, più preferibilmente ha essenzialmente una delle seguenti sequenze amminoacidiche: aa. 5-19 di SEQ ID N.2; aa.50-64 di SEQ ID N.2; aa.5-19 di SEQ ID N.4; aa 50-65 di SEQ ID N.4. In a preferred aspect the peptide molecule is included in the SEQ ID No. 2 or SEQ ID No. 4 sequences, more preferably it has essentially one of the following amino acid sequences: aa. 5-19 of SEQ ID No. 2; aa.50-64 of SEQ ID N.2; aa.5-19 of SEQ ID N.4; aa 50-65 of SEQ ID N.4.
E’ ulteriore oggetto dell’invenzione la molecola peptidica come sopra descritta per uso medico, preferibilmente per il trattamento di malattie mitocondriali. A further object of the invention is the peptide molecule as described above for medical use, preferably for the treatment of mitochondrial diseases.
E’ ulteriore oggetto dell’invenzione una composizione farmaceutica comprendente almeno una molecola peptidica dell’invenzione e opportuni diluenti e/o eccipienti. A further object of the invention is a pharmaceutical composition comprising at least one peptide molecule of the invention and suitable diluents and / or excipients.
E’ ulteriore oggetto dell’invenzione una molecola di acido nucleico codificante per la molecola peptidica dell’invenzione. A further object of the invention is a nucleic acid molecule coding for the peptide molecule of the invention.
E’ ulteriore oggetto dell’invenzione un vettore di espressione comprendente la molecola di acido nucleico come sopra descritta e codificante sotto il controllo di adatte sequenze promotrici e/o regolatrici la molecola peptidica dell’invenzione. A further object of the invention is an expression vector comprising the nucleic acid molecule as described above and encoding the peptide molecule of the invention under the control of suitable promoter and / or regulatory sequences.
Le sequenze amminoacidiche soppressive dei difetti dei mutanti, sono: The mutant defect suppressive amino acid sequences are:
A) l’intera porzione C-Term (66 aminoacidi) del gene codificante per la Leucil-tRNA sintetasi mitocondriale di lievito (NAM2) e suoi frammenti, tra i quali due frammenti di 15 aminoacidi, di seguito sottolineati: Cterm 30_31NAM2 (nt.13-57 di SEQ ID N.1; aa.5-19 di SEQ ID N.2) e Cterm32_33NAM2 (nt.148-192 di SEQ ID N.1; aa. 50-64 di SEQ ID N.2). C Term NAM2 sequenza nucleotidica (SEQ ID N.1) e amminoacidica (SEQ ID N.2) A) the entire C-Term portion (66 amino acids) of the gene encoding yeast mitochondrial leucyl-tRNA synthetase (NAM2) and its fragments, including two fragments of 15 amino acids, underlined below: Cterm 30_31NAM2 (nt .13-57 of SEQ ID N.1; aa.5-19 of SEQ ID N.2) and Cterm32_33NAM2 (nt.148-192 of SEQ ID N.1; aa. 50-64 of SEQ ID N.2) . C Term NAM2 nucleotide (SEQ ID N.1) and amino acid (SEQ ID N.2) sequence
aaa ttc aaa aag ttt caa att gtg gta aat ggc aga gtc aaa ttc atg 48 Lys Phe Lys Lys Phe Gln Ile Val Val Asn Gly Arg Val Lys Phe Met aaa ttc aaa aag ttt caa att gtg gta aat ggc aga gtc aaa ttc atg 48 Lys Phe Lys Lys Phe Gln Ile Val Val Asn Gly Arg Val Lys Phe Met
1 5 10 15 1 5 10 15
tac acg gct gac aaa aac ttt ttg aaa tta ggt agg gat gct gtt att 96 Tyr Thr Ala Asp Lys Asn Phe Leu Lys Leu Gly Arg Asp Ala Val Ile tac acg gct gac aaa aac ttt ttg aaa tta ggt agg gat gct gtt att 96 Tyr Thr Ala Asp Lys Asn Phe Leu Lys Leu Gly Arg Asp Ala Val Ile
20 25 30 20 25 30
gaa act ttg atg aac tta ccg gaa ggg aga atg tat ttg atg aat aaa 144 Glu Thr Leu Met Asn Leu Pro Glu Gly Arg Met Tyr Leu Met Asn Lys gaa act ttg atg aac tta ccg gaa ggg aga atg tat ttg atg aat aaa 144 Glu Thr Leu Met Asn Leu Pro Glu Gly Arg Met Tyr Leu Met Asn Lys
35 40 45 35 40 45
aaa atc aaa aaa ttt gtc atg aaa ttc aat gtg att agt ttc tta ttc 192 Lys Ile Lys Lys Phe Val Met Lys Phe Asn Val Ile Ser Phe Leu Phe aaa atc aaa aaa ttt gtc atg aaa ttc aat gtg att agt ttc tta ttc 192 Lys Ile Lys Lys Phe Val Met Lys Phe Asn Val Ile Ser Phe Leu Phe
50 55 60 50 55 60
cac aag taa 201 His Lys cac aag taa 201 His Lys
65 65
B) l’intera porzione C-Term (67 aminoacidi) del gene codificante per la Leucil-tRNA sintetasi mitocondriale umana (LARS2) e suoi frammenti, tra i quali due frammenti di 15 amminoacidi, di seguito sottolineati: Cterm 30_31LARS2 (nt.13-57 di SEQ ID N.3; aa.5-19 di SEQ ID N.4) e Cterm 32_33LARS2 (nt.148-195 di SEQ ID N.3; aa.50-65 di SEQ ID N. B) the entire C-Term portion (67 amino acids) of the gene encoding human mitochondrial Leucyl-tRNA synthetase (LARS2) and its fragments, including two fragments of 15 amino acids, underlined below: Cterm 30_31LARS2 (nt. 13-57 of SEQ ID N.3; aa.5-19 of SEQ ID N.4) and Cterm 32_33LARS2 (nt.148-195 of SEQ ID N.3; aa.50-65 of SEQ ID N.
4). 4).
C Term LARS2 sequenza nucleotidica (SEQ ID N.3) e amminoacidica (SEQ ID N.4) C Term LARS2 nucleotide sequence (SEQ ID # 3) and amino acid (SEQ ID # 4)
gag gtt gtc cag atg gca gtt ctg atc aac aat aaa gct tgt ggc aaa 48 Glu Val Val Gln Met Ala Val Leu Ile Asn Asn Lys Ala Cys Gly Lys gag gtt gtc cag atg gca gtt ctg atc aac aat aaa gct tgt ggc aaa 48 Glu Val Val Gln Met Ala Val Leu Ile Asn Asn Lys Ala Cys Gly Lys
1 5 10 15 1 5 10 15
att cct gtg ccc caa caa gtt gcc cgg gac cag gac aaa gtc cac gaa 96 Ile Pro Val Pro Gln Gln Val Ala Arg Asp Gln Asp Lys Val His Glu att cct gtg ccc caa caa gtt gcc cgg gac cag gac aaa gtc cac gaa 96 Ile Pro Val Pro Gln Gln Val Ala Arg Asp Gln Asp Lys Val His Glu
20 25 30 20 25 30
ttt gtt ctt caa agc gag ctg ggt gtc agg ctt ttg caa gga cga agc 144 Phe Val Leu Gln Ser Glu Leu Gly Val Arg Leu Leu Gln Gly Arg Ser ttt gtt ctt caa agc gag ctg ggt gtc agg ctt ttg caa gga cga agc 144 Phe Val Leu Gln Ser Glu Leu Gly Val Arg Leu Leu Gln Gly Arg Ser
35 40 45 35 40 45
atc aag aag tcc ttc ctt tcc ccg aga act gcc ctc atc aac ttc ctg 192 Ile Lys Lys Ser Phe Leu Ser Pro Arg Thr Ala Leu Ile Asn Phe Leu atc aag aag tcc ttc ctt tcc ccg aga act gcc ctc atc aac ttc ctg 192 Ile Lys Lys Ser Phe Leu Ser Pro Arg Thr Ala Leu Ile Asn Phe Leu
50 55 60 50 55 60
gtg caa gat tga 207 Val Gln Asp gtg caa gat tga 207 Val Gln Asp
65 65
L’allineamento della sequenza C-Term della leucil-tRNA sintetasi mitocondriale di H. sapiens (prima riga) e S. cerevisiae (seconda riga), ottenuto da http://bioinfo.genotoul.fr/multalin/multalin.html, à ̈ mostrato di seguito; gli aa. in nero indicano identità , in grigio similitudine per carica: The alignment of the C-Term sequence of mitochondrial leucyl-tRNA synthetase of H. sapiens (first line) and S. cerevisiae (second line), obtained from http://bioinfo.genotoul.fr/multalin/multalin.html, It is shown below; aa. in black they indicate identity, in gray similarity for office:
EVVQMAVLINNKACGKIPVPQQVAR-DQDKVHEFVLQSELGVRLLQGRSIKKSFLSPRTALINFLVQD EVVQMAVLINNKACGKIPVPQQVAR-DQDKVHEFVLQSELGVRLLQGRSIKKSFLSPRTALINFLVQD
KFKKFQIVVNGRVKFMYTADKNFLKLGRDAVIETLMNLPEGRMYLMNKKIKKFVM—-KFNVISFLFHK KFKKFQIVVNGRVKFMYTADKNFLKLGRDAVIETLMNLPEGRMYLMNKKIKKFVM⠀ "-KFNVISFLFHK
La presente invenzione sarà ora descritta in sue forme di attuazione esemplificative e non limitative, facendo riferimento alle seguenti figure: The present invention will now be described in exemplary and non-limiting embodiments thereof, with reference to the following figures:
- Fig. 1. (A) Fenotipo di crescita del WT (MCC123, depositato presso DBVPG, Università di Perugia, Italia), del mutante isogenico Val C25T (equivalente alla mutazione umana m. 1624 C→T) e dello stesso mutante trasformato con i vettori multicopia contenenti i geni codificanti per la valil-tRNA sintetasi mit. di S. cerevisiae (Sc) (pVAS1) o di H. sapiens (Hs) (pVARS2), per la leucil-tRNA sintetasi mit di Sc. (pNAM2) o di Hs (pLARS2) e per la istidil-tRNA sintetasi mit di Sc. (pHTS1). Diluizioni seriali sono effettuate su piastre 3% glicerolo e osservate dopo 3 gg di incubazione a 28°C. L’effetto soppressivo à ̈ osservato anche a 37°C. (B) Fenotipo di crescita del WT (MCC123), del mutante isogenico Leu C26(25)T (equivalente alla mutazione umana m. 3256 C>T) e dello stesso mutante trasformato con i vettori con i geni codificanti per Sc mt Leu-RS (pNAM2), Hs mt Leu-RS (pLARS2), Sc mt Val-RS (pVAS1), Hs mt Val-RS (pVARS2) e Sc mt His-RS (pHTS1). Diluizioni seriali sono effettuate su piastre 3% glicerolo e osservate dopo 5 gg di incubazione a 28°C. L’effetto soppressivo à ̈ osservato anche a 37°C. Per il mutante Leu C26(25)T fortemente deficiente per le capacità respiratorie occorre usare diluizioni più basse e un tempo di incubazione più elevato (5 gg) (Montanari et al.2010). - Fig. 1. (A) Growth phenotype of WT (MCC123, deposited at DBVPG, University of Perugia, Italy), of the isogenic mutant Val C25T (equivalent to the human mutation m. 1624 Câ † 'T) and of the same transformed mutant with the multicopy vectors containing the genes encoding the mit valyl-tRNA synthase. of S. cerevisiae (Sc) (pVAS1) or of H. sapiens (Hs) (pVARS2), for the leucyl-tRNA synthetase mit of Sc. (pNAM2) or of Hs (pLARS2) and for the histidyl-tRNA synthetase mit of Sc. (PHTS1). Serial dilutions are carried out on 3% glycerol plates and observed after 3 days of incubation at 28 ° C. The suppressive effect is also observed at 37 ° C. (B) Growth phenotype of WT (MCC123), of the isogenic mutant Leu C26 (25) T (equivalent to the human mutation m. 3256 C> T) and of the same mutant transformed with vectors with genes coding for Sc mt Leu- RS (pNAM2), Hs mt Leu-RS (pLARS2), Sc mt Val-RS (pVAS1), Hs mt Val-RS (pVARS2) and Sc mt His-RS (pHTS1). Serial dilutions are carried out on 3% glycerol plates and observed after 5 days of incubation at 28 ° C. The suppressive effect is also observed at 37 ° C. For the mutant Leu C26 (25) T severely deficient in respiratory capacity it is necessary to use lower dilutions and a longer incubation time (5 days) (Montanari et al.2010).
- Fig. 2. Soppressione del fenotipo difettivo di crescita. A) La crescita in glicerolo del mutante MValC25T à ̈ recuperata in presenza del plasmide multicopia contenente la versione wild-type del gene NAM2 codificante per la leucil-tRNA sintetasi mitocondriale (pNAM2WT) e con il dominio Cterm dello stesso gene (pNAM2CTerm). B) La crescita in glicerolo del mutante MLeu C26(25)T à ̈ recuperata dai tre plasmidi multicopia contenenti il dominio Cterm della leucil-tRNA sintetasi (wild-type,Cterm and ∆CP1) - Fig. 2. Suppression of the growth defective phenotype. A) The growth in glycerol of the MValC25T mutant is recovered in the presence of the multicopy plasmid containing the wild-type version of the NAM2 gene encoding mitochondrial leucyl-tRNA synthetase (pNAM2WT) and with the Cterm domain of the same gene (pNAM2CTerm). B) The growth in glycerol of the MLeu C26 (25) T mutant is recovered from the three multicopy plasmids containing the Cterm domain of the leucyl-tRNA synthase (wild-type, Cterm and ∠† CP1)
- Fig. 3. Diluizioni seriali di due WT (MCC123), del mutante IleT34(33)C e del mutante trasformato con il plasmide multicopia vuoto o contenente il gene NAM2 (codificante per Sc leucil-tRNA sintetasi), gene LARS2 (codificante per Hs leucil-tRNA sintetasi) e la variante LARS230_31Cterm erano piastrate su una piastra contenente 3% glicerolo, 0,1% galattosio e incubate a 37°C per 3 gg. Come controllo, in tutti gli esperimenti delle figure à ̈ stato anche piastrato il mutante trasformato con il solo vettore, senza inserto (empty plasmid). - Fig. 3. Serial dilutions of two WTs (MCC123), of the IleT34 (33) C mutant and of the mutant transformed with the empty multicopy plasmid or containing the NAM2 gene (coding for Sc leucyl-tRNA synthetase), LARS2 gene (coding for Hs leucyl-tRNA synthetase) and the variant LARS230_31Cterm were plated on a plate containing 3% glycerol, 0.1% galactose and incubated at 37 ° C for 3 days. As a control, in all the experiments in the figures, the transformed mutant was also plated with the vector only, without an insert (empty plasmid).
- Fig. 4. Visualizzazione dei domini della Leucil-tRNA sintetasi con la GFP clonata (privata del codone di inizio della traduzione) in fase di lettura al 3’ terminale. - Fig. 4. Visualization of the domains of Leucyl-tRNA synthetase with the cloned GFP (deprived of the translation start codon) being read at the 3â € ™ terminal.
Materiali, metodi e risultati Materials, methods and results
Mezzi di coltura e condizioni di crescita Culture media and growth conditions
I ceppi sono cresciuti in terreno di coltura complete YP (1% estratto di lievito, 1% peptone, Difco) con differenti quantità di glicerolo o glucosio o galattosio addizionati con adenina (45µg/ml). Il terreno minimo era 0.7% “yeast nitrogen base†(Difco), 5% solfato di ammonio e 2% glucosio, con l’aggiunta dei necessari marcatori auxotrofi. Per le piastre solide si aggiunge 1.5% agar (Difco). La capacità di respirare può essere valutata in base alla capacità di crescita su glicerolo, mentre il difetto respiratorio consente solo la crescita su glucosio. Per verificare l’effetto soppressivo delle sequenze che intendiamo brevettare si confronta la capacità di crescere su terreno contenente glicerolo (3%) come unica fonte di carbonio di gocce di una sospensione progressivamente diluita di cellule di lievito wt che respirano e crescono, del mutante (che ha difetti di crescita) e dei trasformanti. Strains grew in complete YP culture medium (1% yeast extract, 1% peptone, Difco) with different amounts of glycerol or glucose or galactose added with adenine (45µg / ml). The minimum medium was 0.7% â € œyeast nitrogen baseâ € (Difco), 5% ammonium sulfate and 2% glucose, with the addition of the necessary auxotrophic markers. For solid plates 1.5% agar (Difco) is added. The ability to breathe can be evaluated based on the growth capacity on glycerol, while the respiratory defect only allows growth on glucose. To verify the suppressive effect of the sequences we intend to patent, we compare the ability of the mutant to grow on medium containing glycerol (3%) as the only source of carbon of drops of a progressively diluted suspension of wt yeast cells that breathe and grow. (which has growth defects) and transformants.
Ceppi e plasmidi Strains and plasmids
Sono stati usati lieviti di Saccharomyces cerevisiae WT MCC123-LSA (Deposito n. 29P del 15_12_2010 in accordo con il Trattato di Budapest presso DBVPG, Università di Perugia, Italia; MAT a, ade2, ura3-52, ∆leu, kar1-1, KanR , rho+), la mutazione rho° à ̈ derivata in seguito a trattamento con Bromuro di Etidio e tre mutanti mitocondriali isogenici synportatori di una mutazione nella posizione 25 del tRNA mitocondriale per leucina (MLeu C26(25)T), per valina (MVal C25T) (Feuermann et al 2003; De Luca et al. 2009, Montanari et al.2010) e per Isoleucina (ile T34(33)). Yeasts of Saccharomyces cerevisiae WT MCC123-LSA were used (Deposit n.29P dated 15_12_2010 in accordance with the Treaty of Budapest at DBVPG, University of Perugia, Italy; MAT a, ade2, ura3-52, ∠† leu, kar1-1 , KanR, rho +), the rho ° mutation is derived following treatment with ethidium bromide and three isogenic mitochondrial mutants carrying a mutation at position 25 of the mitochondrial tRNA for leucine (MLeu C26 (25) T), for valine ( MVal C25T) (Feuermann et al 2003; De Luca et al. 2009, Montanari et al. 2010) and for Isoleucine (ile T34 (33)).
Sequenza wt mt tRNALeu, ID: 854609, tL(UAA)Q (SEQ ID No.7): Sequence wt mt tRNALeu, ID: 854609, tL (UAA) Q (SEQ ID No.7):
T T.
GCTATTTTGGTGGAATTGGTAGACACGATACTCTTAAGATGTATTACTTTACAGTA TGAAGGTTCAAGTCCTTTAAATAGCAATA GCTATTTTGGTGGAATTGGTAGACACGATACTCTTAAGATGTATTACTTTACAGTA TGAAGGTTCAAGTCCTTTAAATAGCAATA
Sequenza wt mt tRNAVal ID: 854626 tV(UAC)Q (SEQ ID No.8): Sequence wt mt tRNAVal ID: 854626 tV (UAC) Q (SEQ ID No.8):
T T.
AGGAGATTAGCTTAATTGGTATAGCATTCGTTTTACACACGAAAGATTATAGGTT CGAACCCTATATTTCCTAAAT AGGAGATTAGCTTAATTGGTATAGCATTCGTTTTACACACGAAAGATTATAGGTT CGAACCCTATATTTCCTAAAT
Sequenza wt mt tRNAIle ID: 854618 tI(GAU)Q (SEQ ID No.9): Sequence wt mt tRNAIle ID: 854618 tI (GAU) Q (SEQ ID No.9):
C GAAACTATAATTCAATTGGTTAGAATAGTATTTTGATAAGGTACAAATATAGGTT CAATCCCTGTTAGTTTCATAT C GAAACTATAATTCAATTGGTTAGAATAGTATTTTGATAAGGTACAAATATATAGGTT CAATCCCTGTTAGTTTCATAT
Le frecce indicano le sostituzioni presenti nei mutanti di lievito nei quali à ̈ stata saggiata l’attività soppressiva da parte dei peptidi dell’invenzione. Tali mutazioni sono equivalenti a mutazioni umane che provocano effetti diversi. La mutazione nel tRNALeu mitocondriale provoca un gravissimo difetto respiratorio che produce nell’uomo una sindrome detta MELAS (Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke) che à ̈ una malattia neurodegenerativa progressiva. La mutazione nel tRNAval mitocondriale provoca nell’uomo la sindrome di Leigh ed encefalomiopatia, una condizione patologica a penetranza variabile e compatibile con l’omoplasmia [R. McFarland, K.M. Clark, A.A.Morris, R.W. Taylor, S. Macphail, R.N. Lightowlers, D.M. Turnbull, Multiple neonatal deaths due to a homoplasmic mitochondrial DNA mutation, Nat. Genet. 30 (2002) 145–146.]. La mutazione del tRNAile mitocondriale ha effetti equivalenti alla sostituzione patologica 4291 T>C che provoca dislipidemia e sindrome metabolica di ipomagnesemia (Wilson, F. H., Hariri, A., Farhi, A., Zhao, H., Petersen, K. F., Toka, H. R., Nelson-Williams, C., Raja, K. M., Kashgarian, M., Shulman, G. I., Scheinman, S. J., Lifton, R. P. (2004) A cluster of metabolic defects caused by mutation in a mitochondrial tRNA Science .306 ,1190-1194) . The arrows indicate the substitutions present in the yeast mutants in which the suppressive activity of the peptides of the invention has been tested. These mutations are equivalent to human mutations that cause different effects. The mutation in the mitochondrial tRNALeu causes a very serious respiratory defect that produces in humans a syndrome called MELAS (Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke) which is a progressive neurodegenerative disease. The mutation in the mitochondrial tRNAval causes in man Leigh's syndrome and encephalomyopathy, a pathological condition with variable penetrance and compatible with homoplasmy [R. McFarland, K.M. Clark, A.A. Morris, R.W. Taylor, S. Macphail, R.N. Lightowlers, D.M. Turnbull, Multiple neonatal deaths due to a homoplasmic mitochondrial DNA mutation, Nat. Genet. 30 (2002) 145â € “146.]. Mitochondrial tRNAile mutation has equivalent effects to the pathological 4291 T> C substitution that causes dyslipidemia and metabolic syndrome of hypomagnesaemia (Wilson, F. H., Hariri, A., Farhi, A., Zhao, H., Petersen, K. F., Toka, H. R. , Nelson-Williams, C., Raja, K. M., Kashgarian, M., Shulman, G. I., Scheinman, S. J., Lifton, R. P. (2004) A cluster of metabolic defects caused by mutation in a mitochondrial tRNA Science .306, 1190-1194 ).
I mutanti di lievito sono ottenuti per trasformazione biolistica bombardando cellule del ceppo MCC123-LSA rho° con microproiettili ad alta velocità in Biolistics PDS-1000 (DuPont Biolistic Particle Delivery System, Wilmington, DE) equipaggiato con un sistema di accelerazione a pressione con eliosystem. I dettagli della trasformazione e la mutagenesi sito specifica sono descritti in H. Rohou, S. Francisci, T. Rinaldi, L. Frontali, M. Bolotin-Fukuhara, Reintroduction of a characterized Mit tRNA glycine mutation into yeast mitochondrial provides a new tool for the study of human neurodegenerative diseases, Yeast 18 (2000) 219–227 e in M. Feuermann, S. Francisci, T. Rinaldi, C. De Luca, H. Rohou, L. Frontali, M. Bolotin- Fukuhara, The yeast counterparts of human †̃MELAS’ mutations causemitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu, EMBO Rep. 4 (2003) 53–58.]. I citoduttanti sono ottenuti per incroci come descritto in De Luca et al. [C. De Luca, Y. Zhou, A. Montanari, V. Morea, R. Oliva, C. Besagni, M. Bolotin- Fukuhara, L. Frontali, S. Francisci, Can yeast be used to study mitochondrial diseases? Biolistic tRNA mutants for the analysis of mechanisms and suppressors, Mitochondrion 9 (2009) 408–41]. Protocolli standard [J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Ito, H., Fukuda, Y., Murata, K., Kimura, A., 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168] sono usati per le trasformazioni di Escherichia coli e lievito così come per le preparazioni dei plasmidi. The yeast mutants are obtained by biolistic transformation by bombarding cells of the MCC123-LSA rho ° strain with high-speed micro-projectiles in Biolistics PDS-1000 (DuPont Biolistic Particle Delivery System, Wilmington, DE) equipped with a pressure acceleration system with heliosystem. The details of the transformation and site-specific mutagenesis are described in H. Rohou, S. Francisci, T. Rinaldi, L. Frontali, M. Bolotin-Fukuhara, Reintroduction of a characterized Mit tRNA glycine mutation into yeast mitochondrial provides a new tool for the study of human neurodegenerative diseases, Yeast 18 (2000) 219â € “227 and in M. Feuermann, S. Francisci, T. Rinaldi, C. De Luca, H. Rohou, L. Frontali, M. Bolotin- Fukuhara, The yeast counterparts of human â € ̃MELASâ € ™ mutations causemitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu, EMBO Rep. 4 (2003) 53â € “58.]. The cytoductants are obtained by crosses as described in De Luca et al. [C. De Luca, Y. Zhou, A. Montanari, V. Morea, R. Oliva, C. Besagni, M. Bolotin- Fukuhara, L. Frontali, S. Francisci, Can yeast be used to study mitochondrial diseases? Biolistic tRNA mutants for the analysis of mechanisms and suppressors, Mitochondrion 9 (2009) 408â € “41]. Standard Protocols [J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Ito, H., Fukuda, Y., Murata, K., Kimura, A., 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163â € “168] are used for the transformations of Escherichia coli and yeast as well as for the preparations of plasmids.
Sovraespressione e soppressione; plasmidi multicopia Overexpression and suppression; multicopy plasmids
I plasmidi multicopia usati per gli esperimenti di soppressione sono i seguenti: The multi-copy plasmids used for the suppression experiments are as follows:
pENAM2 (Prof. C.J. Herbert): la sequenza cromosomale di 4,484 bp che contiene il gene NAM2 (NCBI ID: 851098 ) codificante per la leucil-tRNA sintetasi mitocondriale di lievito e la regione di DNA a monte del gene che contiene il promotore, era clonata nel polilinker del vettore multicopia pFL44S (ATCC Cat. No. 77205) (G.Y. Li, C.J. Herbert, M. Labouesse, P.P. Slonimski, In vitro mutagenesis of the mitochondrial leucyl-tRNA synthetase of S. cerevisiae reveals residues critical for its in vivo activities, Curr. Genet.22 (1992) 69–74.) La sequenza del cromosoma 12, clonata nel plasmide pFL44S, dalle coordinate 881167 -885651 à ̈ la seguente (SEQ ID No. 5; il gene NAM2 à ̈ compreso nelle coordinate 882067-884751). Evidenziati gli inneschi per amplificare il frammento codificante per il C-Terminale: pENAM2 (Prof. C.J. Herbert): the chromosomal sequence of 4,484 bp containing the gene NAM2 (NCBI ID: 851098) encoding the yeast mitochondrial leucyl-tRNA synthetase and the DNA region upstream of the gene containing the promoter, was cloned in the polylinker of the pFL44S multicopy vector (ATCC Cat. No. 77205) (G.Y. Li, C.J. Herbert, M. Labouesse, P.P. Slonimski, In vitro mutagenesis of the mitochondrial leucyl-tRNA synthetase of S. cerevisiae reveals residues critical for its in vivo activities, Curr. Genet.22 (1992) 69â € “74.) The sequence of chromosome 12, cloned in the plasmid pFL44S, from coordinates 881167 -885651 is the following (SEQ ID No. 5; the NAM2 gene is included in the coordinates 882067-884751). The triggers to amplify the coding fragment for the C-Terminal are highlighted:
TGGCGGTCAAAATTGCACTTTTACCACTACCATTATTACCAACAATAAAATTCAGCCTAG AACCAAGCTCAAGTTCAAAATGTTCATGGCACATAAAGTTTCGCAAGATCACTTTCTTAA TATATCCTGAAGGTGACTCTTCCAAAAAGTTATCTTGATCCGCCGTGGCCACGTCAGAGG AGCTCCTAAACCCGCTGTCTTCATCTAGTGTATTGCTGTTAAACTGTGTCATGGGGGCAA ATTGATGACGTCTGCGTTTTCTCTGCTGCTGCTGCTCTTCGTTTTCCTGCTGCGCAGTCA GTGAAAGTAGCTCATCATCAACCTGCTCTATAGGTCTTTTACCACTAATCGTAGTCGAAA TCATACCATTTAGCAATCTAACAAGAATATGCTCTTTATTATTATTAACAGCAGCTACTT ATCTCTTGCGAACCTATATGCCTCTTGTCTTTTTTTCTTATTATGAACTTCACTACATGA AATATAAATATTCATGTTGATAGATCCAAACATTGAAATTTAGTATACGCGACGCGTTTT TGGCAAGTTTAACTTAGAATTTTCACTATAAAACAGAAATTTTGATAAAACTTGGATGAA AATGTTCCTTTTGCCTAACTATCAAATACCACAGAAAAATCTTTAATACCATGCCATACT ACTCCTGGTCTGTTCATGAACTTGCTTGACATTTCCCTACCTCTGACACACTTTTTGATC CAAGAATCTCATTTCTTATCTCCGCGAAGAATCAAAGAAACCGAAGAAAGTCGTTTTGTT GCTCCGGTGGAGAATCAAGAACTTGGAGGAAGGAGCACAGCAGAGTGTTAAACAAGGCGC TAGTACTCTATATTGTAGAGTCTGTACGTGAATAAATCGACGTGTGTGTACGGTGGAAAA ATGCTGTCTCGACCTTCAAGCCGATTCCTATCCACTAAAAGGGGCCCTGGACCTGCAGTG AAAAAATTAATTGCAATTGGGGAGAAATGGAAACAGAAGACAACCCGTGGCTTGCCTAAG CAGGATACTCTGAATAGCGGGTCGAAATATATTCTGTGCCAGTTTCCATATCCTTCTGGG GCGCTTCACATAGGACATCTCCGAGTTTATGTCATTAGCGACTCTTTGAATAGATTTTAC AAACAAAAAGGGTACAACGTGATACATCCGATGGGATGGGATGCTTTTGGGTTACCTGCC GAGAACGCTGCTATAGAAAGAAGCATTAATCCGGCCATATGGACTAGGGACAATATTGCA AAAATGAAACAACAAATGCAAAGTATGCTGGCAAATTTTGATTGGGACAGAGAAATAACT ACATGTGATCCCGAATACTATAAATTCACCCAATGGATTTTCTTAAAACTTTTTGAAAAT GGCTTAGCTTATCGTAAAGAAGCAGAAATTAATTGGGATCCCGTTGATATGACAGTTTTG GCTAATGAACAAGTGGATGCCCAGGGCCGTTCTTGGAGATCAGGGGCTATTGTGGAAAAG AAGCAGCTAAAACAGTGGTTTTTGGGAATAACGAAATTCGCTCCTAAATTGAAAAAGCAC TTGAACCAACTGAAGGACTGGCCTTCCAACGTGAAGCAAATGCAAAAAAATTGGATAGGC GAATCTGTGGGTGCAGAATTAGTGTTCAAAGTTGCGGACCCCAAATTTGAAAACTTGATT GTCTTTACAACAAGACCGGAAACTCTTTTTGCTGTACAGTATGTTGCTCTCGCATTAGAC CACCCAATTGTACAAAAATACTGCGAGGAAATGCCGGATTTAAAAGAGTTTATACAGAAA AGTGATCAATTACCAAACGATACGAAGGAAGGATTTCAGTTACCTAACATAAAGGCCGTA AATCCCTTAACTAAAGAGGAAGTCCCCATATTTGCAGCTCCATATGTGGTCAGCAGCTAT GGTTCAGCACCTAGTGCAGTAATGGGTTGTCCAGGACACGATAACCGAGATTTTGAGTTT TGGCAAACAAATTGTCCTGGTGAACATATCAAGACGTGCATAGCACCTTTTTTTGACGAT GCTTCAAAAGTAACTGAACAGGAAAGACAAAGAATAATTGATACTGTCCCGTTCACATCT ACTGACGGTGTCCTAACCAAAGAATGCGGAGAACACTCAGGAGTTCTCACTGTAGTGGCA AGGAAATCGATAATGGGGATGTTGAATAGCGAAGGACTGTCTAAGAGCGTCGTTAGATAC AAAATTAGAGATTGGCTGATAAGTAGACAAAGATACTGGGGTACTCCAATACCCATCATT CACTGCGACAACTGTGGACCTGTCCCCGTTCCAGAAAGTGATCTACCTGTCAAGCTACCA GAACTTGAAGGCTTGGATACAAAAGGGAATCCGCTGTCCACAATCGATGAATTTGTAAAT GTCGCCTGTCCTTCATGCGGGAGCCCTGCGAAAAGAGAAACTGACACCATGGATACTTTT ATCGATAGTTCTTGGTATTATTTCAGATTCTTGGATCCCAAAAACACTTCAAAACCATTT GATCGTGAAATTGCAAGTAAAAATATGCCGGTTGATATTTATATTGGTGGAGTAGAACAT GCTATCTTACACTTGTTATACTCAAGGTTTATTGCTAAATTTCTCGGATCTATCAATGCA TGGAGCGACCCTGCTGGCATCTTCGAACCATTCAAAAAACTGGTGACACAAGGAATGGTT CAAGGGAAAACCTATGTTGACCCCGATTCTGGTAAATTTTTGAAGCCTGACGAGTTAACC TTTGTAAATGACTCTCCAGATGGCAACACAGTGATTATTAAATCAAATGGCAAGGTTCCG GTAGTCTCTTATGAAAAAATGTCGAAATCTAAATACAATGGTGCAGATCCAAATGAATGT ATTTTAAGACATGGACCTGATGCTACGAGAGCACATATCCTCTTCCAAAGTCCAATTGCA GATGCCCTGAATTGGGATGAATCCAAGATTGTTGGTATAGAACGTTGGTTGCAGAAGGTT CTTCATTTGACCAAAAATATTCTCAGTCTCGAGAAGGATTTGGCAATAAGTAAAGACTAC AAGACCCCGACCGACTTAAATGATGCAGAAGTGAAATTTCACAACGATTTCCAACGCTTC TTGAAATCAATTACGGAATCATTTGAAGTTAATCTATCGTTAAACACTGTAATATCTGAT TATATGAAGCTAACCAATATTTTAGAAAGTGCATTGAAAAAAGGTGAAGTAAGGAATGAA ATGATAGTACAGAATTTACAAAAGTTGGTAACCGTTATATATCCTGCTGTCCCGTCAATT TCAGAAGAAGCTGCAGAGATGATCAACTCCCAAATGGAATGGAACCAATACCGCTGGCCA GAAGTTGAGCGCACTACTGAGTCCAAATTCAAAAAGTTTCAAATTGTGGTAAATGGCAGA GTCAAATTCATGTACACGGCTGACAAAAACTTTTTGAAATTAGGTAGGGATGCTGTTATT GAAACTTTGATGAACTTACCGGAAGGGAGAATGTATTTGATGAATAAAAAAATCAAAAAA TTTGTCATGAAATTCAATGTGATTAGTTTCTTATTCCACAAGTAATCATATGACGCCATG AATTTACCACTAGTGCTTGTAAATAGTTAAATATTAATGCCTTATAGATATAAATTGTCC AATCTCCTTTTTGGAAGTTATAATATATATATATATATATATATGTATATAAATGTGGAT GCATATTCTAAGAATTTTGAACGCTATACTTTGAGAGACTCTTCAAATATGTAAACAAGA ATGCTGCAATGTCTCTATATGGATTAGCAGTGTTACTCAAATGCATTAAAATCTTTACTC TCAAGTCGTGAATGTTATTAACGCCCTTTATCCAACCATGCTGGCCAATCTCATCATGAT GAACTCGAAAGAAATTGTTAAACGTCTCTTCATTTATGATCCCACTGGTGAATTTTATTT TGGTATTTTCGGCCGTTTCCAGTCGTCTTAGAATAATAATACATATGTAGGATAGCGCTG GTATACCAGTTATCGTGAAAAATTTAGCGTTCTTGAAGTCTGCAGTTGGGATATAAAGAT TCTCCAAAATCTGCTTAAAGAAATCTGGTGAGACTCCAAACAAACTTTTGGATGACAGAA CTTTATTCCTGTAAAGATAATTTGTCAAATCCATGATATATTGGTTCTGCATCCGAACGA ATTTGTTTGTTGACGGGTATTGCATCAATTTATTTTTCGTAGCAACCAAGTACCTGCACG CAGCTGATAATAGCAAAGGATCGTCCGAACTGAACACTTTATTCATGATGTCAGGGCCCA CAATCAAATATTGGATCGCATAATCCGACTTCGTTTGTTTCGACAGTTGAGTTAAAATAT CTAACGAGCTGATGAAAGCCATCGTAGAAAATCTGTTTGAAGTAGCATTGTTAAAGATTT TGTCCAAAGTTAGCAGGCTTTGCATGTCATGCAGTAGTGTCAACC TGGCGGTCAAAATTGCACTTTTACCACTACCATTATTACCAACAATAAAATTCAGCCTAG AACCAAGCTCAAGTTCAAAATGTTCATGGCACATAAAGTTTCGCAAGATCACTTTCTTAA TATATCCTGAAGGTGACTCTTCCAAAAAGTTATCTTGATCCGCCGTGGCCACGTCAGAGG AGCTCCTAAACCCGCTGTCTTCATCTAGTGTATTGCTGTTAAACTGTGTCATGGGGGCAA ATTGATGACGTCTGCGTTTTCTCTGCTGCTGCTGCTCTTCGTTTTCCTGCTGCGCAGTCA GTGAAAGTAGCTCATCATCAACCTGCTCTATAGGTCTTTTACCACTAATCGTAGTCGAAA TCATACCATTTAGCAATCTAACAAGAATATGCTCTTTATTATTATTAACAGCAGCTACTT ATCTCTTGCGAACCTATATGCCTCTTGTCTTTTTTTCTTATTATGAACTTCACTACATGA AATATAAATATTCATGTTGATAGATCCAAACATTGAAATTTAGTATACGCGACGCGTTTT TGGCAAGTTTAACTTAGAATTTTCACTATAAAACAGAAATTTTGATAAAACTTGGATGAA AATGTTCCTTTTGCCTAACTATCAAATACCACAGAAAAATCTTTAATACCATGCCATACT ACTCCTGGTCTGTTCATGAACTTGCTTGACATTTCCCTACCTCTGACACACTTTTTGATC CAAGAATCTCATTTCTTATCTCCGCGAAGAATCAAAGAAACCGAAGAAAGTCGTTTTGTT GCTCCGGTGGAGAATCAAGAACTTGGAGGAAGGAGCACAGCAGAGTGTTAAACAAGGCGC TAGTACTCTATATTGTAGAGTCTGTACGTGAATAAATCGACGTGTGTGTACGGTGGAAAA ATGCTGTCTCGACCTTCAAGCCGATTCCTATCCACTAAAAGGGGCCCTGGACCTGCAGTG AAAAAATTAATTGCAATTGGGGAG AAATGGAAACAGAAGACAACCCGTGGCTTGCCTAAG CAGGATACTCTGAATAGCGGGTCGAAATATATTCTGTGCCAGTTTCCATATCCTTCTGGG GCGCTTCACATAGGACATCTCCGAGTTTATGTCATTAGCGACTCTTTGAATAGATTTTAC AAACAAAAAGGGTACAACGTGATACATCCGATGGGATGGGATGCTTTTGGGTTACCTGCC GAGAACGCTGCTATAGAAAGAAGCATTAATCCGGCCATATGGACTAGGGACAATATTGCA AAAATGAAACAACAAATGCAAAGTATGCTGGCAAATTTTGATTGGGACAGAGAAATAACT ACATGTGATCCCGAATACTATAAATTCACCCAATGGATTTTCTTAAAACTTTTTGAAAAT GGCTTAGCTTATCGTAAAGAAGCAGAAATTAATTGGGATCCCGTTGATATGACAGTTTTG GCTAATGAACAAGTGGATGCCCAGGGCCGTTCTTGGAGATCAGGGGCTATTGTGGAAAAG AAGCAGCTAAAACAGTGGTTTTTGGGAATAACGAAATTCGCTCCTAAATTGAAAAAGCAC TTGAACCAACTGAAGGACTGGCCTTCCAACGTGAAGCAAATGCAAAAAAATTGGATAGGC GAATCTGTGGGTGCAGAATTAGTGTTCAAAGTTGCGGACCCCAAATTTGAAAACTTGATT GTCTTTACAACAAGACCGGAAACTCTTTTTGCTGTACAGTATGTTGCTCTCGCATTAGAC CACCCAATTGTACAAAAATACTGCGAGGAAATGCCGGATTTAAAAGAGTTTATACAGAAA AGTGATCAATTACCAAACGATACGAAGGAAGGATTTCAGTTACCTAACATAAAGGCCGTA AATCCCTTAACTAAAGAGGAAGTCCCCATATTTGCAGCTCCATATGTGGTCAGCAGCTAT GGTTCAGCACCTAGTGCAGTAATGGGTTGTCCAGGACACGATAACCGA GATTTTGAGTTT TGGCAAACAAATTGTCCTGGTGAACATATCAAGACGTGCATAGCACCTTTTTTTGACGAT GCTTCAAAAGTAACTGAACAGGAAAGACAAAGAATAATTGATACTGTCCCGTTCACATCT ACTGACGGTGTCCTAACCAAAGAATGCGGAGAACACTCAGGAGTTCTCACTGTAGTGGCA AGGAAATCGATAATGGGGATGTTGAATAGCGAAGGACTGTCTAAGAGCGTCGTTAGATAC AAAATTAGAGATTGGCTGATAAGTAGACAAAGATACTGGGGTACTCCAATACCCATCATT CACTGCGACAACTGTGGACCTGTCCCCGTTCCAGAAAGTGATCTACCTGTCAAGCTACCA GAACTTGAAGGCTTGGATACAAAAGGGAATCCGCTGTCCACAATCGATGAATTTGTAAAT GTCGCCTGTCCTTCATGCGGGAGCCCTGCGAAAAGAGAAACTGACACCATGGATACTTTT ATCGATAGTTCTTGGTATTATTTCAGATTCTTGGATCCCAAAAACACTTCAAAACCATTT GATCGTGAAATTGCAAGTAAAAATATGCCGGTTGATATTTATATTGGTGGAGTAGAACAT GCTATCTTACACTTGTTATACTCAAGGTTTATTGCTAAATTTCTCGGATCTATCAATGCA TGGAGCGACCCTGCTGGCATCTTCGAACCATTCAAAAAACTGGTGACACAAGGAATGGTT CAAGGGAAAACCTATGTTGACCCCGATTCTGGTAAATTTTTGAAGCCTGACGAGTTAACC TTTGTAAATGACTCTCCAGATGGCAACACAGTGATTATTAAATCAAATGGCAAGGTTCCG GTAGTCTCTTATGAAAAAATGTCGAAATCTAAATACAATGGTGCAGATCCAAATGAATGT ATTTTAAGACATGGACCTGATGCTACGAGAGCACATATCCTCTTCCAAAGTCCAATTGCA GATGCCCTGAA TTGGGATGAATCCAAGATTGTTGGTATAGAACGTTGGTTGCAGAAGGTT CTTCATTTGACCAAAAATATTCTCAGTCTCGAGAAGGATTTGGCAATAAGTAAAGACTAC AAGACCCCGACCGACTTAAATGATGCAGAAGTGAAATTTCACAACGATTTCCAACGCTTC TTGAAATCAATTACGGAATCATTTGAAGTTAATCTATCGTTAAACACTGTAATATCTGAT TATATGAAGCTAACCAATATTTTAGAAAGTGCATTGAAAAAAGGTGAAGTAAGGAATGAA ATGATAGTACAGAATTTACAAAAGTTGGTAACCGTTATATATCCTGCTGTCCCGTCAATT TCAGAAGAAGCTGCAGAGATGATCAACTCCCAAATGGAATGGAACCAATACCGCTGGCCA GAAGTTGAGCGCACTACTGAGTCCAAATTCAAAAAGTTTCAAATTGTGGTAAATGGCAGA GTCAAATTCATGTACACGGCTGACAAAAACTTTTTGAAATTAGGTAGGGATGCTGTTATT GAAACTTTGATGAACTTACCGGAAGGGAGAATGTATTTGATGAATAAAAAAATCAAAAAA TTTGTCATGAAATTCAATGTGATTAGTTTCTTATTCCACAAGTAATCATATGACGCCATG AATTTACCACTAGTGCTTGTAAATAGTTAAATATTAATGCCTTATAGATATAAATTGTCC AATCTCCTTTTTGGAAGTTATAATATATATATATATATATATATGTATATAAATGTGGAT GCATATTCTAAGAATTTTGAACGCTATACTTTGAGAGACTCTTCAAATATGTAAACAAGA ATGCTGCAATGTCTCTATATGGATTAGCAGTGTTACTCAAATGCATTAAAATCTTTACTC TCAAGTCGTGAATGTTATTAACGCCCTTTATCCAACCATGCTGGCCAATCTCATCATGAT GAACTCGAAAGAAATTGTTAAACGTCTCTTCATTT ATGATCCCACTGGTGAATTTTATTT TGGTATTTTCGGCCGTTTCCAGTCGTCTTAGAATAATAATACATATGTAGGATAGCGCTG GTATACCAGTTATCGTGAAAAATTTAGCGTTCTTGAAGTCTGCAGTTGGGATATAAAGAT TCTCCAAAATCTGCTTAAAGAAATCTGGTGAGACTCCAAACAAACTTTTGGATGACAGAA CTTTATTCCTGTAAAGATAATTTGTCAAATCCATGATATATTGGTTCTGCATCCGAACGA ATTTGTTTGTTGACGGGTATTGCATCAATTTATTTTTCGTAGCAACCAAGTACCTGCACG CAGCTGATAATAGCAAAGGATCGTCCGAACTGAACACTTTATTCATGATGTCAGGGCCCA CAATCAAATATTGGATCGCATAATCCGACTTCGTTTGTTTCGACAGTTGAGTTAAAATAT CTAACGAGCTGATGAAAGCCATCGTAGAAAATCTGTTTGAAGTAGCATTGTTAAAGATTT TGTCCAAAGTTAGCAGGCTTTGCATGTCATGCAGTAGTGTCAACC
Le diversi varianti dei geni NAM2 o LARS2 erano clonate nel vettore pYES2.1/V5- The different variants of the NAM2 or LARS2 genes were cloned into the pYES2.1 / V5- vector
His-TOPO (pYES2.1TOPO TA Expression Kit, Invitrogen) sotto il controllo del promotore His-TOPO (pYES2.1TOPO TA Expression Kit, Invitrogen) under the control of the promoter
inducibile Gal1, come segue. inducible Gal1, as follows.
Per ottenere il plasmide pNAM2C-term, il DNA genomico dal ceppo selvatico MCC123-LSA To obtain the pNAM2C-term plasmid, genomic DNA from the wild MCC123-LSA strain
era amplificato con gli inneschi: was amplified with the triggers:
NamCtermEcoRI+ GGAATTCCGATGAAATTCAAAAAGTTTCAAAT (SEQ ID No.10) NamCtermEcoRI + GGAATTCCGATGAAATTCAAAAAGTTTCAAAT (SEQ ID No.10)
NamCtermXbaI- GCTCTAGAGCTTACTTGTGGAATAAGAAAC (SEQ ID No.11). NamCtermXbaI- GCTCTAGAGCTTACTTGTGGAATAAGAAAC (SEQ ID No.11).
In grassetto i siti di restrizione e sottolineati i codoni di inizio e fine della traduzione. Il The restriction sites are bold and the translation start and end codons are underlined. The
frammento amplificato di 220 bp, contenente la sequenza codificante per il C-Term di NAM2 amplified fragment of 220 bp, containing the coding sequence for the C-Term of NAM2
era clonato e la presenza dei frammenti di digestione EcoRI/XbaI e PvuII/XbaI erano utilizzati per confermare la presenza e l’orientamento del frammento clonato nel plasmide. Veniva effettuato il sequenziamento per verifica. was cloned and the presence of the EcoRI / XbaI and PvuII / XbaI digestion fragments were used to confirm the presence and orientation of the cloned fragment in the plasmid. Sequencing was performed for verification.
Le varianti C-Term leucil-tRNA sintetasi umana erano subclonate dal plasmide pLARS2 (pYES2 Invitrogen+LARS2 ID: 23395 (Montanari et al 2010)). The human C-Term leucyl-tRNA synthetase variants were subcloned by the plasmid pLARS2 (pYES2 Invitrogen + LARS2 ID: 23395 (Montanari et al 2010)).
Il plasmide pLARS2Cterm30_31 era ottenuto usando gli oligomeri complementari CtermLARS30.31E/B+ (SEQ ID No.12) CGGAATTCCGatggcagttctgatcaacaataaagcttgtggcaaaattcctgtgtgaCGGGATCCCG, e CtermLARS30.31E/B- (SEQ ID No.13) CGGGATCCCGtcacacaggaattttgccacaagctttattgttgatcagaactgccatCGGAATTCCG. Il plasmide pLARS2Cterm30_31 era ottenuto usando gli oligomeri complementari CtermLARS30.31E/B+ (SEQ ID No.12) CGGAATTCCGatggcagttctgatcaacaataaagcttgtggcaaaattcctgtgtgaCGGGATCCCG, e CtermLARS30.31E/B- (SEQ ID No.13) CGGGATCCCGtcacacaggaattttgccacaagctttattgttgatcagaactgccatCGGAATTCCG.
Dopo ibridazione (30 secondi a 95°C, ghiaccio per 1 minuto, 30 secondi a 95°C e 10 minuti a 65°C), la sequenza oligomerica a doppio filamento era ligata. Una digestione con BamHI era effettuata per controllo. After hybridization (30 seconds at 95 ° C, ice for 1 minute, 30 seconds at 95 ° C and 10 minutes at 65 ° C), the double-stranded oligomeric sequence was ligated. A digestion with BamHI was carried out for control.
Il plasmide pLARS2Cterm32_33 era ottenuto usando la stessa procedura di cui sopra, con gli oligomeri complementari: The pLARS2Cterm32_33 plasmid was obtained using the same procedure as above, with the complementary oligomers:
CtermLARS32.33E/B+ (SEQ ID No.14) CGGAATTCCGatgaagaagtccttcctttccccgagaactgccctcatcaacttcctggtgtgaCGGGATCCCG CtermLARS32.33E/B- (SEQ ID No.15) CGGGATCCCGtcacaccaggaagttgatgagggcagttctcggggaaaggaaggacttcttcatCGGAATTCCG. CtermLARS32.33E / B + (SEQ ID No.14) CGGAATTCCGatgaagaagtccttcctttccccgagaactgccctcatcaacttcctggtgtgaCGGGATCCCG CtermLARS32.33E / B- (SEQ ID No.15) CGGttGATCCCGtcacacaggc
Veniva effettuato il sequenziamento per verifica, con gli inneschi forniti con il plasmide del kit commerciale (Invitrogen). Sequencing for verification was performed, with primers supplied with the commercial kit plasmid (Invitrogen).
Localizzazione ed espressione Localization and expression
Per localizzare i prodotti nella cellula, si ottenevano plasmidi con le sequenze fuse con la proteina fluorescente verde GFP di Aequorea Victoria (S65T). To localize the products in the cell, plasmids were obtained with the sequences fused with the green fluorescent protein GFP of Aequorea Victoria (S65T).
Le cellule dei mutanti del tRNALeu e del tRNAval erano trasformate e si otteneva conferma che le sequenze clonate nei vettori pNAM2GFP e pNAM2CtermGFP erano funzionali. La microscopia confocale mostra una localizzazione mitocondriale uniforme (confermata anche per colorazione DAPI). Per contrasto il prodotto del costrutto pNAM2CP1GFP, plasmide che contiene la sequenza codificante per il dominio CP1 della proteina NAM2 coinvolto nello splicing e nell’editing, come controllo negativo (si veda Fig. 4) mostrava una localizzazione diffusa. I risultati sono consistenti con l’effetto soppressivo. The tRNALeu and tRNAval mutant cells were transformed and it was confirmed that the cloned sequences in the pNAM2GFP and pNAM2CtermGFP vectors were functional. Confocal microscopy shows uniform mitochondrial localization (also confirmed by DAPI staining). By contrast, the product of the pNAM2CP1GFP construct, plasmid containing the coding sequence for the CP1 domain of the NAM2 protein involved in splicing and editing, as a negative control (see Fig. 4) showed a diffuse localization. The results are consistent with the suppressive effect.
Per ottenere il plasmide pNAM2GFP, DNA genomico dal YEAST GFP CLONE gene YLR382C (Invitrogen), in cui il gene GFP à ̈ clonato (privato del codone di inizio della traduzione) in fase di lettura al 3’ terminale del gene NAM2 (privato del codone di stop della traduzione) era amplificato con inneschi: To obtain the pNAM2GFP plasmid, genomic DNA from the YEAST GFP CLONE gene YLR382C (Invitrogen), in which the GFP gene is cloned (deprived of the translation start codon) being read at the 3â € ™ terminal of the NAM2 gene (deprived of the translation stop codon) was amplified with triggers:
NAM4+ (SEQ ID No.16) GTCTCGAGAAGGATTTGGCAATAAG e NAM4 + (SEQ ID No.16) GTCTCGAGAAGGATTTGGCAATAAG e
GFPXbaI- (SEQ ID No.17) GCTCTAGAGCTTATTTGTATAGTTCATCC. GFPXbaI- (SEQ ID No.17) GCTCTAGAGCTTATTTGTATAGTTCATCC.
I due geni erano separati da un linker (AGGGGTCGACGGATCCCCGGGTTTAATTAAC SEQ ID No. 18) per facilitarne il sub clonaggio. Il frammento di circa 1,400 bp amplificato era digerito con XbaI e XhoI e ligato al plasmide pNAM2∆Cterm, già digerito e defosforilato con fosfatasi alcalina CIP (Biolabs). Una digestione con XhoI era effettuata per controllo. Per ottenere il plasmide pNAM2CtermGFP un frammento di circa 1,000 bp di DNA genomico era amplificato da YEAST GFP CLONE gene YLR382C (Invitrogen) con inneschi NAMCtermEcoRI+ (SEQ ID No.19) CGGAATTCCGATGAAATTCAAAAAGTTTCAAAT e The two genes were separated by a linker (AGGGGTCGACGGATCCCCGGGTTTAATTAAC SEQ ID No. 18) to facilitate sub-cloning. The approximately 1,400 bp amplified fragment was digested with XbaI and XhoI and ligated to the pNAM2∠† Cterm plasmid, already digested and dephosphorylated with CIP alkaline phosphatase (Biolabs). A digestion with XhoI was carried out for control. To obtain the pNAM2CtermGFP plasmid an approximately 1,000 bp fragment of genomic DNA was amplified by YEAST GFP CLONE gene YLR382C (Invitrogen) with NAMCtermEcoRI + primers (SEQ ID No.19) CGGAATTCCGATGAAATTCAAAAAGTTCAAAT and
GFPXbaI- (SEQ ID No.20) GCTCTAGAGCTTATTTGTATAGTTCATCC. GFPXbaI- (SEQ ID No.20) GCTCTAGAGCTTATTTGTATAGTTCATCC.
Una digestione con EcoRI/XbaI era effettuata per controllo. A digestion with EcoRI / XbaI was carried out for control.
Per ottenere il plasmide pNAM2CP1GFP, un frammento di circa 1,000 bp di DNA genomico selvatico era amplificato da inneschi To obtain the pNAM2CP1GFP plasmid, an approximately 1,000 bp fragment of wild genomic DNA was amplified by primers
EcoRIATGCP1+ (SEQ ID No.21) GGAATTCCATGATAACTACATGTGATC EcoRIATGCP1 + (SEQ ID No.21) GGAATTCCATGATAACTACATGTGATC
e CP1SmaI- (SEQ ID No.22) TCCCCCGGGGGAGAGAACTCCTGAGTGTTC. and CP1SmaI- (SEQ ID No.22) TCCCCCGGGGGAGAGAACTCCTGAGTGTTC.
Dopo digestione con EcoRI / SmaI, il frammento era ligato al plasmide pNAM2∆CtermGFP, già digerito e defosforilato con fosfatasi alcalina CIP (Biolabs). Una digestione con EcoRI/ SmaI era effettuata per controllo. After digestion with EcoRI / SmaI, the fragment was ligated to the plasmid pNAM2∠† CtermGFP, already digested and dephosphorylated with CIP alkaline phosphatase (Biolabs). A digestion with EcoRI / SmaI was carried out for control.
Risultati Results
La Fig. 1 confronta la crescita in glicerolo del ceppo WT con quella dei due mutanti MLeu C26(25)T e MVal C25T, con diversa capacità respiratoria; i difetti respiratori di entrambi i mutanti sono alleviati (soppressi) dalla sovraespressione delle leucil e valil-RS mitocondriali umane e di lievito. La sovraespressione à ̈ ottenuta trasformando le cellule del mutante di lievito con plasmidi multi copia contenenti i geni NAM2 ID: 851098, VAS1 ID: 2543603, LARS2 ID: 23395, VARS2 ID: 57176 . Come controllo negativo à ̈ stato utilizzato il gene HTS1 ID: 856145 (His-RS) (Montanari et al 2010). Fig. 1 compares the growth in glycerol of the WT strain with that of the two mutants MLeu C26 (25) T and MVal C25T, with different respiratory capacity; the respiratory defects of both mutants are alleviated (suppressed) by the overexpression of human and yeast mitochondrial leucyl and valyl-RS. Overexpression is achieved by transforming yeast mutant cells with multi-copy plasmids containing NAM2 ID: 851098, VAS1 ID: 2543603, LARS2 ID: 23395, VARS2 ID: 57176 genes. The HTS1 gene ID: 856145 (His-RS) (Montanari et al 2010) was used as a negative control.
Nella Fig. 2 à ̈ dimostrato l’effetto soppressivo della leucil-RS mitocondriale di lievito (pNAM2) e di diversi costrutti da essa derivati.(pCTerm, pdeltaCterm). In Fig. 2 the suppressive effect of the mitochondrial leucil-RS of yeast (pNAM2) and of various constructs derived from it is demonstrated (pCTerm, pdeltaCterm).
Nella Fig 3 à ̈ riportato l’effetto soppressivo di pNAM2, di pLARS2 (leucil-RS umana) e della variante del C-Ter del gene LARS2 (pLARS2Cterm30_31) provata sul mutante del tRNA IleT34(33)C. La sequenza di 15 aa sovraespressa nel mutante ile T34(33)C sopprime il difetto di crescita in terreno contenente glicerolo come unica fonte di carbonio. Fig 3 shows the suppressive effect of pNAM2, pLARS2 (human leucil-RS) and the C-Ter variant of the LARS2 gene (pLARS2Cterm30_31) tested on the IleT34 (33) C tRNA mutant. The 15 aa sequence overexpressed in the ile T34 (33) C mutant suppresses the growth defect in medium containing glycerol as the sole carbon source.
L’effettiva espressione e localizzazione del prodotto genico, può essere controllata mediante l’inserimento in fase, al terminale 3’, del gene della GFP. The actual expression and localization of the gene product can be controlled by phasing in the GFP gene at the 3â € ™ terminal.
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| Publication number | Publication date |
|---|---|
| IT1406405B1 (en) | 2014-02-21 |
| WO2012089570A1 (en) | 2012-07-05 |
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