Name: expression analysis of mammalian linker-histone subtypes
Uploaded: 2012-03-19T12:00:00
Description: Watch this Scientific Journal Video about Expression Analysis of Mammalian Linker-histone Subtypes at JoVE.com

Dette arbeidet støttes av NIH stipend GM085261 og Georgia Cancer Coalition Distinguished Scholar Award (til YF).

1. Prøveopparbeidelse og RNA Utvinning <ol><li> Før RNA ekstraksjon, bør alle som arbeider overflater og pipetter tørkes med 70% etanol og behandles med RNase dekontaminering løsning, som RNase Zap. Denne praksisen reduserer sjansene for RNase forurensning og RNA degradering. Bruk hansker for alle prosedyrer. </li><li> Hvis du vil trekke RNA fra mus vev, dissekere organ interesse fra avlives mus, og vaske vevet i iskalde Fosfatbufret saltvann (PBS: 0,13 M NaCl, 5 mm Sodium Dinatriumhydrogenfosfat heptahydrat, 5 m...

<ol>
	<li>Fan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H1+linker+histones+are+essential+for+mouse+development+and+affect+nucleosome+spacing+in+vivo.">H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo.</a> Mol. Cell Biol. 23, 4559-4572 (2003).</li><li>Woodcock, C. L., Skoultchi, A. I., Fan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+linker+histone+in+chromatin+structure+and+function:+H1+stoichiometry+and+nucleosome+repeat+length.">Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length.</a> Chromosome Res. 14, 17-25 (2006).</li><li>Shen, X., Gorovsky, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linker+histone+H1+regulates+specific+gene+expression+but+not+global+transcription+in+vivo.">Linker histone H1 regulates specific gene expression but not global transcription in vivo.</a> Cell. 86, 475-483 (1996).</li><li>Alami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+linker-histone+subtypes+differentially+affect+gene+expression+in+vivo.">Mammalian linker-histone subtypes differentially affect gene expression in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 5920-5925 (2003).</li><li>Happel, N., Doenecke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+and+its+isoforms:+contribution+to+chromatin+structure+and+function.">Histone H1 and its isoforms: contribution to chromatin structure and function.</a> Gene. 431, 1-12 (2009).</li><li>Clausell, J., Happel, N., Hale, T. K., Doenecke, D., Beato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+subtypes+differentially+modulate+chromatin+condensation+without+preventing+ATP-dependent+remodeling+by+SWI&#47;SNF+or+NURF.">Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI&#47;SNF or NURF.</a> PLoS One. 4, 0007243-0007243 (2009).</li><li>Khadake, J. R., Rao, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-+chromatin-condensing+properties+of+rat+testes+H1a+and+H1t+compared+to+those+of+rat+liver+H1bdec;+H1t+is+a+poor+condenser+of+chromatin.">DNA- chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec; H1t is a poor condenser of chromatin.</a> Biochemistry. 34, 15792-15801 (1995).</li><li>Orrego, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+affinity+of+mammalian+histone+H1+somatic+subtypes+for+DNA+and+chromatin.">Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin.</a> BMC Biol. 5, 22-22 (2007).</li><li>Th'ng, J. P., Sung, R., Ye, M., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H1+family+histones+in+the+nucleus.+Control+of+binding+and+localization+by+the+C-terminal+domain.">H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain.</a> J. Biol. Chem. 280, 27809-27814 (2005).</li><li>Wang, Z. F., Sirotkin, A. M., Buchold, G. M., Skoultchi, A. I., Marzluff, W. 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+mouse+histone+H1+genes:+gene+organization+and+differential+regulation.">The mouse histone H1 genes: gene organization and differential regulation.</a> J. Mol. Biol. 271, 124-138 (1997).</li><li>Brown, D. T., Sittman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+through+overexpression+and+tagging+of+the+variant+type+of+the+mouse+H1e+and+H1c+genes.">Identification through overexpression and tagging of the variant type of the mouse H1e and H1c genes.</a> J. Biol. Chem. 268, 713-718 (1993).</li><li>Fan, Y., Sirotkin, A., Russell, R. G., Ayala, J., Skoultchi, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+somatic+H1+subtypes+are+dispensable+for+mouse+development+even+in+mice+lacking+the+H1(0)+replacement+subtype.">Individual somatic H1 subtypes are dispensable for mouse development even in mice lacking the H1(0) replacement subtype.</a> Mol. Cell. Biol. 21, 7933-7943 (2001).</li><li>Fan, Y., Skoultchi, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+H1+linker+histone+subtypes+and+their+functions+in+mice.">Genetic analysis of H1 linker histone subtypes and their functions in mice.</a> Methods Enzymol. 377, 85-107 (2004).</li><li>Heid, C. A., Stevens, J., Livak, K. J., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+quantitative+PCR.">Real time quantitative PCR.</a> Genome Res. 6, 986-994 (1996).</li></ol>

Settet av analysene presenteres her gjør omfattende analyse av uttrykket nivåer av pattedyr dennes histone subtyper. Riktig utformet QRT-PCR-analyser gir svært følsomme og nøyaktige målinger av RNA meldinger fra noen pattedyr histone H1 gener. Den kritiske delen av Qrt-PCR-analyser for dennes histone SubType gener er utarbeidelsen av cDNA ved hjelp tilfeldig primer basert revers transkripsjon. mRNA av de fleste histone gener, inkludert de fleste H1 gener, inneholder ikke en lang poly-A hale presentert i andre cell...

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expression analysis of mammalian linker-histone subtypes

Linker histone H1 binder seg til nucleosome kjernen partikkel og linker DNA, tilrettelegging bretting av kromatin til høyere orden struktur. H1 er avgjørende for pattedyr utvikling 1 og regulerer spesifikke genuttrykk in vivo 2-4. Blant de svært bevart histonproteiner, er familien til H1 dennes histoner den mest heterogene gruppen. Det er 11 H1 subtyper i pattedyr som er ulikt regulert under utvikling og i ulike celletyper. Disse H1 subtyper omfatter fem somatiske H1S (H1A-e), utskifting H1 0, 4 bakterie celle spesifikke H1 subtyper, og H1x 5. Tilstedeværelsen av flere H1 undertyper som varierer i DNA bindende affinitet og kromatin komprimering evne 6-9 gir et ekstra nivå av modulering av kromatin funksjon. Dermed er kvantitativ uttrykk analyse av individuelle H1 subtyper, både av mRNA og proteiner, er nødvendig for bedre forståelse av reguleringen av høyereFor kromatinstruktur og funksjon. Her beskriver vi et sett av metoder utviklet for å analysere uttrykket nivåer av enkelte H1 undertyper (figur 1). mRNA uttrykk av ulike H1 variant gener måles ved et sett av svært sensitive og kvantitativ revers transkripsjon-PCR (QRT-PCR) analyser, som er raskere, mer nøyaktig og krever mye mindre prøver sammenlignet med alternativ tilnærming av utrykt. I motsetning til de fleste andre cellulære mRNA meldinger, mRNA for det meste histone gener, deriblant de fleste H1 gener, mangler en lang Polya hale, men inneholder en stilk-løkke struktur på 3 &#39;uoversatt region (UTR) 10. Derfor er cDNAs forberedt fra total RNA ved revers transkripsjon bruke tilfeldige primere stedet for oligo-DT primere. Realtime PCR analyser med primere spesifikke for hver H1 subtypene (tabell 1) er utført for å oppnå svært kvantitativ måling av mRNA nivåer av enkelte H1 subtypes. Uttrykk av rengjøringstjenester gener analyseres som kontroller for normalisering. Den relative overflod av proteiner til hvert H1 subtype og kjernen histoner oppnås gjennom omvendt fase med høy ytelse væskekromatografi (RP-HPLC) analyse av totalt histoner hentet fra mammalske celler 11-13. HPLC-metoden og eluering forholdene beskrevet her gi optimale separasjoner av mus H1 subtyper. Ved å kvantifisere HPLC profilen, beregner vi den relative andelen av individuelle H1 subtyper innenfor H1 familie, samt bestemme H1 til nucleosome ratio i cellene.

Watch this Scientific Journal Video about Expression Analysis of Mammalian Linker-histone Subtypes at JoVE.com

Expression Analysis of Mammalian Linker-histone Subtypes

Vi beskriver et sett av metoder for å analysere uttrykk nivåer av H1 dennes histoner. mRNA av individuelle H1 gener kvantitativt måles ved tilfeldig primer basert revers transkripsjon etterfulgt av real-time PCR, mens protein kvantifisering av H1 histoner oppnås ved HPLC analyse.

Expression Analyse av pattedyrs Linker-histone Subtyper

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

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