تم دعم هذا المنشور من قبل برنامج دعم مشاريع البحث والابتكار التكنولوجي (PAPIIT) ، UNAM [IN223623 إلى XP-M]. UPD هو زميل CONAHCYT (CVU: 883299). نود أن نشكر الدكتور أريان ميندوزا مارتينيز على المساعدة الفنية في صور المجهر الضوئي. تراخيص Bionder: DU26OMVLUU (الشكل 2) ؛ BK26TH9GXH (الشكل 3) ؛ GD26TH80R5 (الشكل 4) ؛ PU26THARYD (الشكل 7) ؛ ML26THAIFG (الشكل 9).

ملاحظة: نوصي بإجراء ستة تحويلات لكل بنية لأن كفاءة تحويل الميتوكوندريا عادة ما تكون منخفضة. ويبين الجدول 2 تكوين وسائط النمو المختلفة.
1. إعداد جزيئات التنغستن
<ol><li>تزن 30 ملغ من جزيئات التنغستن 0.7 ميكرومتر (WPs ، الناقلات الدقيقة) في أنبوب صغير. أضف 1.5 مل من 70٪ ...

التحول الحيوي لميتوكوندريا الخميرة للتحليل الوراثي للطفرات

<ol>
	<li>Bonnefoy, N., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+mutated+initiation+codons+in+the+mitochondrial+COX2+gene+of+Saccharomyces+cerevisiae+fused+to+the+reporter+gene+ARG8m+reveals+lack+of+downstream+reinitiation.">In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation.</a> Mol Gen Genet. 262 (6), 1036-1046 (2000).</li><li>Franco, L. V. R., Su, C. H., McStay, G. P., Yu, G. J., Tzagoloff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cox2p+of+yeast+cytochrome+oxidase+assembles+as+a+stand-alone+subunit+with+the+Cox1p+and+Cox3p+modules.">Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules.</a> J Biol Chem. 293 (43), 16899-16911 (2018).</li><li>Flores-Mireles, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytochrome+b+carboxyl+terminal+region+is+necessary+for+mitochondrial+complex+III+assembly.">The cytochrome b carboxyl terminal region is necessary for mitochondrial complex III assembly.</a> Life Sci Alliance. 6 (7), (2023).</li><li>Rubalcava-Gracia, D., V&#225;zquez-Acevedo, M., Funes, S., P&#233;rez-Mart&#237;nez, X., Gonz&#225;lez-Halphen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+versus+nuclear+gene+expression+and+membrane+protein+assembly:+the+case+of+subunit+2+of+yeast+cytochrome.">Mitochondrial versus nuclear gene expression and membrane protein assembly: the case of subunit 2 of yeast cytochrome.</a> Mol Biol Cell. 29 (7), 820-833 (2018).</li><li>Garc&#237;a-Villegas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cox1+C-terminal+domain+is+a+central+regulator+of+cytochrome+c+oxidase+biogenesis+in+yeast+mitochondria.">The Cox1 C-terminal domain is a central regulator of cytochrome c oxidase biogenesis in yeast mitochondria.</a> J Biol Chem. 292 (26), 10912-10925 (2017).</li><li>Rak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+cells+lacking+the+mitochondrial+gene+encoding+the+ATP+synthase+subunit+6+exhibit+a+selective+loss+of+complex+IV+and+unusual+mitochondrial+morphology.">Yeast cells lacking the mitochondrial gene encoding the ATP synthase subunit 6 exhibit a selective loss of complex IV and unusual mitochondrial morphology.</a> J Biol Chem. 282 (15), 10853-10864 (2007).</li><li>Perez-Martinez, X., Broadley, S. A., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mss51p+promotes+mitochondrial+Cox1p+synthesis+and+interacts+with+newly+synthesized+Cox1p.">Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p.</a> EMBO J. 22 (21), 5951-5961 (2003).</li><li>Sanchirico, M. E., Fox, T. D., Mason, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+mitochondrially+synthesized+Saccharomyces+cerevisiae+Cox2p+and+Cox3p+depends+on+targeting+information+in+untranslated+portions+of+their+mRNAs.">Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs.</a> EMBO J. 17 (19), 5796-5804 (1998).</li><li>Saracco, S. A., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cox18p+is+required+for+export+of+the+mitochondrially+encoded+Saccharomyces+cerevisiae+Cox2p+C-tail+and+interacts+with+Pnt1p+and+Mss2p+in+the+inner+membrane.">Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail and interacts with Pnt1p and Mss2p in the inner membrane.</a> Mol Biol Cell. 13 (4), 1122-1131 (2002).</li><li>McStay, G. P., Su, C. H., Thomas, S. M., Xu, J. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allotopic+expression+of+COX6+elucidates+Atco-driven+co-assembly+of+cytochrome+oxidase+and+ATP+synthase.">Allotopic expression of COX6 elucidates Atco-driven co-assembly of cytochrome oxidase and ATP synthase.</a> Life Sci Alliance. 6 (11), e202301965 (2023).</li><li>Mireau, H., Arnal, N., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Barstar+as+a+selectable+marker+in+yeast+mitochondria.">Expression of Barstar as a selectable marker in yeast mitochondria.</a> Mol Genet Genomics. 270 (1), 1-8 (2003).</li><li>Cohen, J. S., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+green+fluorescent+protein+from+a+recoded+gene+inserted+into+Saccharomyces+cerevisiae+mitochondrial+DNA.">Expression of green fluorescent protein from a recoded gene inserted into Saccharomyces cerevisiae mitochondrial DNA.</a> Mitochondrion. 1 (2), 181-189 (2001).</li><li>Suhm, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+system+to+monitor+mitochondrial+translation+in+yeast.">A novel system to monitor mitochondrial translation in yeast.</a> Microb Cell. 5 (3), 158-164 (2018).</li><li>Rzepka, M., Suhm, T., Ott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+reporter+genes+into+mitochondrial+DNA+in+budding+yeast.">Incorporation of reporter genes into mitochondrial DNA in budding yeast.</a> STAR Protoc. 3 (2), 101359 (2022).</li><li>Steele, D. F., Butler, C. A., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+recoded+nuclear+gene+inserted+into+yeast+mitochondrial+DNA+is+limited+by+mRNA-specific+translational+activation.">Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation.</a> Proc Natl Acad Sci U S A. 93 (11), 5253-5257 (1996).</li><li>Flores-Mireles, D., Camacho-Villasana, Y., P&#233;rez-Mart&#237;nez, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ARG8m+reporter+for+the+study+of+yeast+mitochondrial+translation.">The ARG8m reporter for the study of yeast mitochondrial translation.</a> Methods Mol Biol. 2661, 281-301 (2023).</li><li>Perez-Martinez, X., Butler, C. A., Shingu-Vazquez, M., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+functions+of+Mss51+couple+synthesis+of+Cox1+to+assembly+of+cytochrome+c+oxidase+in+Saccharomyces+cerevisiae+mitochondria.">Dual functions of Mss51 couple synthesis of Cox1 to assembly of cytochrome c oxidase in Saccharomyces cerevisiae mitochondria.</a> Mol Biol Cell. 20 (20), 4371-4380 (2009).</li><li>Bonnefoy, N., Remacle, C., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Saccharomyces+cerevisiae+and+Chlamydomonas+reinhardtii+mitochondria.">Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria.</a> Methods Cell Biol. 80, 525-548 (2007).</li><li>Veloso Ribeiro Franco, L., Barros, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Saccharomyces+cerevisiae+mitochondria+using+the+biolistic+gun.">Transformation of Saccharomyces cerevisiae mitochondria using the biolistic gun.</a> Methods Enzymol. 264, 265-278 (1996).</li><li>Bonnefoy, N., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Saccharomyces+cerevisiae+mitochondria.">Genetic transformation of Saccharomyces cerevisiae mitochondria.</a> Methods Cell Biol. 65, 381-396 (2001).</li><li>Shing&#250;-V&#225;zquez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carboxyl-terminal+end+of+Cox1+is+required+for+feedback+assembly+regulation+of+Cox1+synthesis+in+Saccharomyces+cerevisiae+mitochondria.">The carboxyl-terminal end of Cox1 is required for feedback assembly regulation of Cox1 synthesis in Saccharomyces cerevisiae mitochondria.</a> J Biol Chem. 285 (45), 34382-34389 (2010).</li><li>Bonnefoy, N., Bsat, N., Fox, T. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoduction+as+a+new+tool+in+studying+the+cytoplasmic+heredity+in+yeast.">Cytoduction as a new tool in studying the cytoplasmic heredity in yeast.</a> Mol Cell Biochem. 14 (1-3), 15-18 (1977).</li><li>Conde, J., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutant+of+Saccharomyces+cerevisiae+defective+for+nuclear+fusion.">A mutant of Saccharomyces cerevisiae defective for nuclear fusion.</a> Proc Natl Acad Sci U S A. 73 (10), 3651-3655 (1976).</li><li>Burke, D., Dawson, D., Stearns, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harbor Laboratory. Methods in yeast genetics : a Cold Spring Harbor Laboratory manual. 2000 ed. , (2000).</li><li>Dunham, M. J., Gartenberg, M. R., Brown, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+:+a."> : a.</a> Methods in yeast genetics and genomics : a Cold Spring Harbor Laboratory course manual. 2015 edition. , (2015).</li><li>Ding, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+27+An+improved+method+for+introducing+point+mutations+into+the+mitochondrial+cytochrome+B+gene+to+facilitate+studying+the+role+of+cytochrome+B+in+the+formation+of+reactive+oxygen+species.">Chapter 27 An improved method for introducing point mutations into the mitochondrial cytochrome B gene to facilitate studying the role of cytochrome B in the formation of reactive oxygen species.</a> Methods Enzymol. 456, 491-506 (2009).</li><li>Klein, C. J. L., Olsson, L., Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+control+in+Saccharomyces+cerevisiae:+the+role+of+Mig1+in+metabolic+functions.">Glucose control in Saccharomyces cerevisiae: the role of Mig1 in metabolic functions.</a> Microbiology (Reading). 144, 13-24 (1998).</li><li>Rolland, F., Winderickx, J., Thevelein, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-sensing+and+-signalling+mechanisms+in+yeast.">Glucose-sensing and -signalling mechanisms in yeast.</a> FEMS Yeast Res. 2 (2), 183-201 (2002).</li><li>Gruschke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cbp3-Cbp6+complex+coordinates+cytochrome+b+synthesis+with+bc(1)+complex+assembly+in+yeast+mitochondria.">The Cbp3-Cbp6 complex coordinates cytochrome b synthesis with bc(1) complex assembly in yeast mitochondria.</a> J Cell Biol. 199 (1), 137-150 (2012).</li><li>Seshadri, S. R., Banarjee, C., Barros, M. H., Fontanesi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translational+activator+Sov1+coordinates+mitochondrial+gene+expression+with+mitoribosome+biogenesis.">The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.</a> Nucleic Acids Res. 48 (12), 6759-6774 (2020).</li><li>Rak, M., Tzagoloff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F1-dependent+translation+of+mitochondrially+encoded+Atp6p+and+Atp8p+subunits+of+yeast+ATP+synthase.">F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase.</a> Proc Natl Acad Sci U S A. 106 (44), 18509-18514 (2009).</li><li>He, S., Fox, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+a+yeast+mitochondrial+inner+membrane+protein,+pnt1p,+block+export+of+a+mitochondrially+synthesized+fusion+protein+from+the+matrix.">Mutations affecting a yeast mitochondrial inner membrane protein, pnt1p, block export of a mitochondrially synthesized fusion protein from the matrix.</a> Mol Cell Biol. 19 (10), 6598-6607 (1999).</li><li>Rak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mitochondrial+translation+of+the+ATP8/ATP6+mRNA+by+Smt1p.">Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p.</a> Mol Biol Cell. 27 (6), 919-929 (2016).</li><li>Barrera-Paez, J. D., Moraes, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+genome+engineering+coming-of-age.">Mitochondrial genome engineering coming-of-age.</a> Trends Genet. 38 (8), 869-880 (2022).</li></ol>

ليس لدى المؤلفين أي تضارب في المصالح للكشف عنه.

وصف العمل الحالي كيفية تحويل الميتوكوندريا من الخميرة S. cerevisiae بنجاح. تستغرق العملية ، من قصف المقذوفات الدقيقة عالية السرعة حتى تنقية سلالة الخميرة المقصودة ، ~ 8-12 أسبوعا ، اعتمادا على عدد جولات تنقية سلالة الرو الاصطناعية اللازمة. بعض الخطوات الحاسمة للطريقة هي كما يلي. أولا ، ...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL pipette tips</td>
			<td>Axygen</td>
			<td>T-1000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microtube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L pipette tips</td>
			<td>Axygen</td>
			<td>T-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical bottom tube&#160;</td>
			<td>Axygen</td>
			<td>SCT-25ML-25-S</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>Axygen</td>
			<td>T-200-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical bottom&#160; tube</td>
			<td>Axygen</td>
			<td>SCT-50ML-25-S</td>
			<td></td>
		</tr>
		<tr>
			<td>AfiII</td>
			<td>New England BioLabs</td>
			<td>R0520S</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>SeaKem</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytic balance</td>
			<td>OHAUS</td>
			<td>ARA520</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>TOMY</td>
			<td>ES-315</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolisitic Macrocarrier holder&#160;</td>
			<td>BIO-RAD</td>
			<td>1652322</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>VWR</td>
			<td>89038-528</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -ADE</td>
			<td>Formedium</td>
			<td>DCS0049</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -ARG</td>
			<td>Formedium</td>
			<td>DCS0059</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -LEU</td>
			<td>Formedium</td>
			<td>DCS0099</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -URA</td>
			<td>Formedium</td>
			<td>DCS0169</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture glass flask</td>
			<td>KIMAX KIMBLE</td>
			<td>25615</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture glass tube</td>
			<td>Pyrex</td>
			<td>9820</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>BD</td>
			<td>215520</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>JT Baker&#160;</td>
			<td>9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Millipore</td>
			<td>620006</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>Z265926</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass handle</td>
			<td>Sigma</td>
			<td>S4647</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>JT BAKER</td>
			<td>2136-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Helium tank grade 5 (99.99 %)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>HSTaq&#160; Kit</td>
			<td>PCR BIO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifugue</td>
			<td>Eppendorf</td>
			<td>022620100</td>
			<td></td>
		</tr>
		<tr>
			<td>NdeI</td>
			<td>New England BioLabs</td>
			<td>R0111L</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>New Brunswick scientific</td>
			<td>NB-G25</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PDS-1000/He TM Biolistic Particle Delivery System</td>
			<td>BIO-RAD</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (100X10)</td>
			<td>BD</td>
			<td>252777</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Raffinose</td>
			<td>Formedium</td>
			<td>RAF03</td>
			<td></td>
		</tr>
		<tr>
			<td>Replica plater</td>
			<td>Scienceware</td>
			<td>Z363391</td>
			<td></td>
		</tr>
		<tr>
			<td>Rupture discs 1350 Psi</td>
			<td>BIO-RAD</td>
			<td>1652330</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma</td>
			<td>S7547</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Thermo Scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Rotator</td>
			<td>Thermo Scientific</td>
			<td>88882015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten microcarriers M10</td>
			<td>BIO-RAD</td>
			<td>1652266</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccum pump of 100L/min capacity</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Velvet pads</td>
			<td>Bel-Art</td>
			<td>H37848-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex&#160;</td>
			<td>Scientifc Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Wood aplicator stick</td>
			<td>PROMA</td>
			<td>1820060</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen base without aminoacids</td>
			<td>BD</td>
			<td>291920</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial transformation in baker&#39;s yeast to study translation and respiratory complex assembly

تم استخدام خميرة بيكر Saccharomyces cerevisiae على نطاق واسع لفهم بيولوجيا الميتوكوندريا لعقود. قدم هذا النموذج المعرفة حول مسارات الميتوكوندريا الأساسية والمحفوظة بين حقيقيات النوى والفطريات أو المسارات الخاصة بالخميرة. واحدة من القدرات العديدة ل S. cerevisiae هي القدرة على التعامل مع جينوم الميتوكوندريا ، وهو أمر ممكن حتى الآن فقط في S. cerevisiae والطحالب أحادية الخلية Chlamydomonas reinhardtii. يسمح لنا التحول البيولوجي لميتوكوندريا الخميرة بإدخال طفرات موجهة للموقع ، وإعادة ترتيب الجينات ، وتقديم المراسلين. تستخدم هذه الأساليب بشكل أساسي لفهم آليات عمليتين منسقتين للغاية في الميتوكوندريا: الترجمة بواسطة mitoribosomes وتجميع المجمعات التنفسية و ATP synthase. ومع ذلك ، يمكن استخدام تحويل الميتوكوندريا لدراسة مسارات أخرى. في العمل الحالي ، نوضح كيفية تحويل ميتوكوندريا الخميرة عن طريق قصف المقذوفات الدقيقة عالية السرعة ، واختيار وتنقية المحول المقصود ، وإدخال الطفرة المطلوبة في جينوم الميتوكوندريا.

The yeast Saccharomyces cerevisiae is a widely recognized model used to study mitochondrial biogenesis. Since yeast is an anaerobic, facultative organism, it is possible to extensively study the causes and consequences of introducing mutations that impair respiration. In addition, this organism possesses friendly genetic and biochemical tools to study mitochondrial pathways. However, one of the most powerful resources to explore the mechanisms of respiratory complex assembly and mitochondrial protein synthesis is the ability to transform mitochondria and modify the organelle&#39;s genome. Previously, it has been helpful to introduce in the mitochondrial DNA (mtDNA) point mutations or small deletions/insertions1,2,3,4,5, delete genes6,7, make gene rearrangements7,8, add epitopes to mitochondrial proteins9,10, relocate genes from the nucleus to the mitochondria11,12, and introduce reporter genes like BarStar13, GFP14,15, luciferase16, and the most widely used ARG8m17,18. Mitochondrial genome modifications have allowed us to dissect and identify mechanisms that otherwise would have been difficult to comprehend. For example, the ARG8m reporter gene inserted at the COX1 locus&#160;in the mitochondrial DNA was crucial to understanding that Mss51 has a dual role in Cox1 biogenesis. First, it is a translational activator of the COX1 mRNA, and second, it is an assembly chaperone for the newly made Cox1 protein7,19. This work presents a detailed method to transform S. cerevisiae mitochondria. Although the mitochondrial transformation protocol was published earlier16,20,21,22,23, a visual approach through a video is essential to thoroughly understand the different stages and details of the method. The method consists of various steps and is divided into four general stages:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66856/66856fig01v2.jpg" /> 
Figure 1: Overview of the mitochondrial transformation procedure by high-velocity microprojectile bombardment: 1) The tungsten particles are sterilized and prepared before DNA coating. 2) Two different plasmids are precipitated on the surface of the WPs. One is a bacterial plasmid containing the construct that will be directed to the mitochondrial matrix. The other is a nuclear yeast expression vector carrying an auxotrophy marker. 3) A receptor yeast strain lacking mitochondrial DNA (rho0) is grown on a fermentative carbon source like galactose or raffinose, which does not exert any glucose repression of mitochondrial gene expression34,35. The culture is spread on petri dishes containing the bombardment medium. 4) WPs coated with plasmids are shot to the receptor strain by high-velocity microprojectile bombardment. 5) Positive synthetic rho- colonies containing the mitochondrial plasmid are selected by mating to a tester strain. 6) The mitochondrial construct is integrated into the mitochondrial genome at the desired locus by mating the synthetic rho- strain (donor) with the acceptor strain by a process known as cytoduction. 7) The positive receptor, haploid strain carrying the mitochondrial construct is selected and purified in different media. Abbreviations: WPs = tungsten particles; mtDNA = mitochondrial DNA. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Transformation of cells with the DNA construct intended to integrate into the mitochondrial genome (Figure 1, steps 1-4) 
Although it is possible to directly transform a yeast containing a complete mitochondrial genome (rho+), the transformation is 10-20x more efficient if the cells lack mitochondrial DNA (rho0)24. Tungsten particles (WPs) are coated with two different plasmids that will be co-transformed. The first one is a yeast 2 &#181; expression vector carrying an auxotrophy marker, such as LEU2 or URA3 (e.g., YEp351 or YEp352, respectively). If the biolistic introduction of DNA into the yeast cells is successful, the transformants will grow on the auxotrophy medium (i.e., a medium lacking leucine or uracil). This plasmid is helpful in making the first selection of the cells that acquired the nuclear plasmid; otherwise, the number of resulting colonies would saturate the plate. The second plasmid is a bacterial plasmid (such as pBluescript or similar) containing the mitochondrial construction intended to be integrated into the mitochondrial genome. The construct must contain at least 100 nt of 5&#39; and 3&#39; flanking mitochondrial sequences for recombination with the mitochondrial region of interest. In our experience, larger flanking sequences are more likely to successfully recombine with the target locus in the mitochondrial genome.
A specific example is shown in Figure 225. In this example, the aim was to delete the region of the mitochondrial COX1 gene coding for the last 15 amino acids of the corresponding protein (Cox1&#916;C15) (Figure 2A). The bacterial plasmid carrying the COX1&#916;C15 mutation contained 395 nt and 990 nt of the gene&#39;s 5&#39; and 3&#39; untranslated regions, respectively. The plasmid was derived from pBluescript (pXPM61) and, together with the 2 &#181; plasmid YEp352, they were coprecipitated on the surface of WPs (Figure 2B). The WPs were then introduced by microprojectile bombardment into the selected recipient strain (Figure 2C). This strain, named NAB6926, is a MATa, rho0 strain bearing the kar1-1 and ade2 alleles&#160;(the importance of these characteristics is discussed below). The cells were plated on bombardment media lacking uracil to select those that acquired the 2 &#181; plasmid (Figure 2C). Cells were grown for 5-7 nights at 30 &#176;C.
Selection of the cells that acquired the bacterial plasmid containing the mitochondrial construct and the nuclear 2 &#181; plasmid carrying the auxotrophy marker (Figure 1, step 5) 
The positive colonies will maintain many copies of the bacterial plasmid in their mitochondria22. Since the transformed cells are originally rho0, no mitochondrial sequences support the translation of the mitochondrial construct present in the plasmid; hence, the transformed mitochondrial gene will not be expressed. It is necessary to mate the transformants with a tester strain to detect the yeast colonies that acquired the mitochondrial plasmid. The mitochondrial genome of the tester strain contains a nonfunctional mutated version of the gene of interest. After mating, the mitochondria will fuse, and the mitochondrial sequence included in the transformed plasmid will recombine with the mutated mitochondrial gene from the tester strain; consequently, the recovery of the WT gene will reconstitute function. The resulting diploid will have a detectable positive phenotype (usually the capacity to grow in respiratory medium or a medium lacking arginine). The positive cells carrying the bacterial plasmid in mitochondria are named &#34;synthetic rho- cells&#34;. In the specific example of Figure 2D,&#160;synthetic rho- cells carrying the COX1&#916;C15 construct (named XPM199) were replica-plated on a medium lacking uracil (this is the master plate from which positive colonies were purified). They were also replica-plated on a tester strain L4527 lawn, which contains the nonfunctional mutation cox1D369N. After mating for two nights, the mitochondrial genome of L45 and XPM199 recombined, resulting in a functional COX1 gene; therefore, the diploids recovered the ability to grow on respiratory media. From the master plate, we picked the positive colonies. They were streaked on plates lacking uracil, and the selective tests were repeated to obtain pure synthetic rho- cells (Figure 2E). It is important to note that the testing plate is only for the identification of synthetic rho- colonies, and mutants cannot be recovered from these plates.
Integration of the construct into the mitochondrial genome of the intended strain 
This step is achieved by a process called cytoduction28,29 (Figure 1, step 6). In this approach, the synthetic rho- strain (donor strain) is mated to the intended acceptor strain of the opposite mating type. An essential requirement is that at least one of the two mating strains carries the kar1-1 mutation to impede nuclear fusion30. Hence, mating of the two cells produces a binucleated zygote (Figure 3). The mitochondrial network from the parental cells (donor and acceptor) fuse and the mitochondrial DNA molecules recombine. The binucleated zygotes are incubated/recovered to allow budding, which will produce haploid cells. These haploids carry the nuclear background of one of the parental cells. Similarly, haploids can carry the mitochondrial DNA from either the parental cell or the recombined mitochondrial DNA of interest. However, the kar1-1 mutation is not 100% effective, and some true diploids will be formed during the mating28,29. The synthetic rho- strain (donor, XPM199) carried the kar1-1 allele in the example. As a selective marker, it had the ade2 allele, making it an auxotroph for adenine (Figure 4). The acceptor strain was XPM10, a rho+ strain bearing the cox1&#916;::ARG8m&#160;construct, where the reporter ARG8m&#160;replaced the COX1 codons in the mitochondrial genome7. The mixture of both cell cultures was added to a YPD plate as a drop to allow mating. After 3-5 h, the cells were observed under a light microscope to detect the formation of shmoos (Figure 3B), a cell shape associated with mating. After the incubation/recovery time, the binuclear zygotes were plated on a medium lacking adenine to prevent the growth of the donor cells.
Selection of the haploid strain carrying the mitochondrial genome of interest (Figure 1, step 7) 
A mixture of donor, acceptor, and diploid cells is present after cytoduction. Therefore, after the incubation/recovery of the binucleated zygotes, cells must be grown in different selective media to identify and purify the haploids of interest. The selective media depends on the genotypes of the donor, acceptor strains, and the intended mitochondrial construction. However, in general, the reasoning for choosing the selective media is: i) after incubation/recovery, the cytoduction mix is grown on selective medium where the donor parental strain cannot grow. In the specific example in Figure 4A,B, the donor (synthetic rho- strain) carries the nonfunctional ade2 allele. Thus, the cytoduction mix must be incubated on a medium plate lacking adenine. This is the master plate. ii) Once the colonies grow, the master plate is replicated again on a medium lacking adenine (to generate a new master plate from where the positive colonies of interest will be purified), and next, on a medium where only diploids can grow. This is necessary to avoid further inadvertent purification of diploid colonies. In the case of the example from Figure 4C, only diploids can grow on media lacking leucine. iii) The master plate is also replicated on media where only those acceptor haploids that incorporated the mitochondrial DNA of interest will grow. In the example of Figure 4C,&#160;haploids grow on respiratory media containing ethanol/glycerol as a carbon source since the Cox1&#916;C15 protein is functional. The resulting rho+ strain bearing the mtDNA of interest was named XPM20925. The strains used in Figure&#160;2 and Figure 4 are listed in Table 1.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66856/66856fig02.jpg" /> 
Figure 2: Diagram describing a specific example of mitochondrial transformation and selection of positive rho- cells. (A) The intended modification of the mitochondrial genome was a deletion of the region coding for the last 15 amino acids of the Cox1 subunit (Cox1&#916;C15). (B) WPs were coated with two plasmids to transform yeast cells. One was the 2 &#181; plasmid Yep352 that was directed and expressed in the nucleus. The other was plasmid pXPM61, which contains the COX1&#916;C15 allele plus 395 nt and 990 nt of the COX1 5&#39; and 3&#39;-UTRs, respectively. (C) The WPs were introduced into cells from the strain NAB69, which lacks mitochondrial DNA (rho0 strain). Transformant colonies obtained from the bombardment plates were replicated on a medium lacking uracil, the auxotrophic marker of the nuclear plasmid YEp352. (D) To select the positive rho- colonies, the -URA plate was replicated on a media lacking uracil. This was the master plate from which the positive colonies were picked and isolated. It was also replicated on plates with rich media (YPD) and a lawn of a tester strain. During mating, the synthetic rho- mitochondrial DNA was recombined with the mitochondrial DNA from the tester strain, L4527, which carries a mutation in the COX1 gene (D369N). The diploids that grew on respiratory media contained the transformed DNA. The corresponding colonies were picked from the master plate to restreak and purify. To help identify the positive colonies in the master plate throughout all replica plating, marks were made with a permanent marker on the edges of the plates (green lines). (E) The selected positive colonies were restreaked on a medium lacking uracil. This was the new master plate. As in D, two more rounds of testing were carried out to purify the synthetic rho- colonies. Abbreviations: WPs = tungsten particles; mtDNA = mitochondrial DNA; -URA/Sorb = lacking uracil/ containing Sorbitol;&#160;WT = wild type. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66856/66856fig03v2.jpg" /> 
Figure 3: Diagram describing the cytoduction procedure. (A) General overview of how cells mate during cytoduction.&#160;During cytoduction, mitochondria from the donor and acceptor strains fuse so mitochondrial DNA from both strains recombine. Since nuclear fusion is reduced due to the kar1-1 mutation30,&#160;binuclear zygotes are formed. After some incubation/recovery time, the binuclear zygotes bud and haploid acceptors containing the intended mitochondrial mutation are selected. (B) Mating of the synthetic rho- strain (donor) and the acceptor strain through cytoduction allows the formation of shmoos (red arrows), a characteristic shape of mating yeast. The image was taken under a light microscope with a 100x objective. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66856/66856fig04.jpg" /> 
Figure 4: Diagram describing a specific example of cytoduction to integrate the mutation COX1&#916;C15 in the mitochondrial genome. (A) Liquid cultures of the synthetic rho- strain (donor, XPM199) and the strain of interest (acceptor, XPM10) were mixed to mate. After shmoo formation, the mix was recovered in a liquid culture for 2-4 h. Next, the cytoduction mix was spread on a plate with a medium lacking adenine to prevent the growth of the donor cells. (B) Schematic representation of the recombination events of the mitochondrial DNA from the donor (XPM199) and the acceptor (XPM10) during cytoduction. (C) The master plate was replicated on different selective media to identify and purify those haploids that integrated the intended mitochondrial gene in the organelle&#39;s DNA (XPM209)25. Abbreviations: -ADE = lacking adenine; -LEU = lacking leucine. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

تسليط الضوء على المؤلف: تطوير التقنيات والاكتشافات في تخليق البروتين وتجميعه من خلال أبحاث الميتوكوندريا المبتكرة

تحول الميتوكوندريا في بيكر

Our research explores the regulation of mitochondrial protein synthesis and its connection to inner membrane assembly, crucial for oxidative phosphorylation. Using the model, we study the molecular mechanism of mitochondrial function, leveraging its suitability for detailed molecular and genetic investigations. Many technologies are used for research in our field, like RNA deep sequencing, multiomic technologies, cryo-electron microscopy, high-velocity bombardment to transform gene mitochondria, among others.We have made important contributions to understand the role and mechanisms of action of many proteins involved in mitochondrial translation regulation and on respiratory complexes III and IV assembly. This is the case for proteins like Mss51, Pet309, Pet54, mS38, CPP3, CPP6, among others. This protocol is an indispensable tool for understanding how mitoribosomes find the STARD-CHOL mitochondrial mRNAs.It has been the key to understanding the role of specific nucleotides of mitochondrial mRNAs in translation regulation. These are some examples of what this technique can address. The only available resource to mitochondrial sequences and to use translation of the in the mitochondrial DNA is our protocol, high-velocity bombardment transformation of gene mitochondria.This method has provided invaluable information regarding translation and respiratory complexes assembly that otherwise we could not obtain.

يقدم هذا القسم بعض النتائج التمثيلية من المراحل المختلفة لتحول الميتوكوندريا. يوضح الشكل 6 إجراء القصف. حملت خلايا الرو الاصطناعية بلازميدا بكتيريا مع الجين المراسل ARG8m ، والذي سيحل محل تسلسل الترميز لجين الميتوكوندريا (الشكل 6 أ). بعد القص?...

يشرح هذا العمل كيفية تحويل ميتوكوندريا الخميرة باستخدام طريقة بيولوجية. نوضح أيضا كيفية اختيار وتنقية المحولات وكيفية إدخال الطفرة المطلوبة في الموضع المستهدف داخل جينوم الميتوكوندريا.

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about ...

mitochondrial-transformation-bakers-yeast-to-study-translation

Research

JoVE Journal

Genetics

Mitochondrial Transformation in Baker&#39;s Yeast to Study Translation and Respiratory Complex Assembly

405 Views.  Universidad Nacional Autónoma de México. Our research explores the regulation of mitochondrial protein synthesis and its connection to inner membrane assembly, crucial for oxidative phosphorylation. Using the model, we study the molecular mechanism of mitochondrial function, leveraging its suitability for detailed molecular and genetic investigations. Many technologies are used for research in our field, like RNA deep sequencing, multiomic technologies, cryo-electron microscopy, high-velocity bombardment to transform gene mitochondr...