[C-terminal domain of saccharomyces cerevisiae protein ChI4 binds to centromere DNA fragment of yeast chromosome III].
We have expressed in Escherichia coli a recombinant protein consisting of N-terminal peptide omega 10s10 (11 aa) fused with part (aa 135-458) of yeast protein Chl4 involved in the chromosome segregation in Saccharomyces cerevisiae. Mice were immunized with the antigen purified from inclusion bodies, and a polyclonal serum against yeast protein Chl4 was raised. MW of the detected yeast protein Chl4 was approximately 54 kDa, corresponding to the full length ORF translation. C-terminal portion of Chl4 (aa 376-458), containing Helin-Turn-Helix (HTH) motif of DNA-binding, was fused in frame after E. coli maltose binding protein MalE. The soluble fusion was affinity purified using an alternative procedure on the preswollen amylose column. This protein and a 32P labelled 620 bp fragment of yeast CEN3 DNA were used in the DNA-mobility shift assay in polyacrylamide and agarose gels. The binding was detected in the presence and absence of Zn2+ ions. The data obtained could support participation of Chl4 in a direct binding to the yeast centromere in the CBF complex. The result is in a certain agreement with the data on photocrosslinking proteins of the CBF3 complex with the centromere DNA, where the minor protein with a molecular weight of 55-55 kDa was also detected (Espelin C. W. et al., 1997. J. Cell Biol. 139: 1383-1396).
Shmelev
A V
AV
Institute of Cytology, Russian Academy of Sciences, St. Petersburg. anton.shmelev@helsinki.fi
Koriabin
M Iu
MIu
Davydenko
S G
SG
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism*; D002503:Centromere / Q000235:genetics / Q000378:metabolism; D015825:Chromosomes, Fungal; D004271:DNA, Fungal / Q000378:metabolism*; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C081874:CHL4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins