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Anti-FLAG-affinity purification of Umbrea associated factors

ABSTRACT: Anti-FLAG-affinity purification of Umbrea associated factors from nuclear extracts of Drosophila Schneider cells expressing FLAG-HA-Umbrea. Anti-FLAG -affinity purifications from Schneider cell nuclear extracts not expressing any FLAG-tagged protein served as a control SDS-PAGE separting both samples, 9 slices per sample, reduction and alkylation, digestion with trypsin. LC-MS on a Ultimate3000-LTQ Orbitrap XL Raw data were analysed using the MaxQuant 1.2.2.5 software package. Identified proteins were considered as interaction partners if their MaxQuant intensities displayed a greater than 10 fold enrichment compared to control anti-FLAG purifications from L2-4 nuclear extracts not expressing any FLAG-tagged protein

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Andreas Thomae

LAB HEAD: Andreas Thomae

PROVIDER: PXD000163 | Pride | 2013-10-03

REPOSITORIES: Pride

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			Action	DRS
	Ross_et_al_txt.zip	Other
	SID1127_Ctrl-IP_1.RAW	Raw
	SID1127_Ctrl-IP_2.RAW	Raw
	SID1127_Ctrl-IP_3.RAW	Raw
	SID1127_Ctrl-IP_4.RAW	Raw

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Publications

Stepwise evolution of essential centromere function in a Drosophila neogene.

Ross Benjamin D BD Rosin Leah L Thomae Andreas W AW Hiatt Mary Alice MA Vermaak Danielle D de la Cruz Aida Flor A AF Imhof Axel A Mellone Barbara G BG Malik Harmit S HS

Science (New York, N.Y.) 20130601 6137

Evolutionarily young genes that serve essential functions represent a paradox; they must perform a function that either was not required until after their birth or was redundant with another gene. How young genes rapidly acquire essential function is largely unknown. We traced the evolutionary steps by which the Drosophila gene Umbrea acquired an essential role in chromosome segregation in D. melanogaster since the gene's origin less than 15 million years ago. Umbrea neofunctionalization occurre ...[more]

PMID: 23744945

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A pair of centromeric proteins mediates reproductive isolation in Drosophila species

Project description:Speciation involves the reproductive isolation of natural populations due to the sterility or lethality of their hybrids. However, the molecular basis of hybrid lethality and the evolutionary driving forces that provoke it, remain largely elusive. The hybrid male rescue (Hmr) and the lethal hybrid rescue (Lhr) genes serve as a model to study speciation in Drosophilids as their interaction causes lethality in male hybrid offspring. Here we show that HMR and LHR form a centromeric complex necessary for proper chromosome segregation. We find that the Hmr expression level is substantially higher in D. melanogaster whereas Lhr expression levels are increased in D. simulans. The resulting elevated amount of HMR/LHR complex in hybrids results in an extensive mislocalisation of the complex, an interference with the regulation of transposable elements and an impairment of cell proliferation. Our findings provide evidence for a major role of centromere divergence in the generation of biodiversity. Raw data were analysed using the MaxQuant 1.2.2.5 software package. Identified proteins were considered as interation partners if their MaxQuant iBAQ values displayed a greater than 16fold enrichment compared to control anti-FLAG purifications from Schneider cell nuclear extracts not expressing any FLAG-tagged protein.

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