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9206 Delair Wy. Elk Grove, CA 95616 (916) 479 4544 [email protected] Ron M. G. Menorca • Pak Kwong • Ravi Maruthachalam • Simon Chan College of Biological Sciences: Section of Plant Biology University of California Davis, 95616 Faithful chromosome segregation is mediated by the centromere and the kinetochore (1). The main protein that recruits other kinetochore proteins and assembles a functional centromere is the centromere-specific histone H3 (CENH3) which replaces the canonical histone H3 in centromeric chromatin (2). Conventional histone H3 is highly conserved due to its importance in DNA packaging. In contrast, the CENH3 primary sequence has been shown to evolve rapidly. We hypothesize that there is a conserved structure that underlies CENH3 function, despite the lack of sequence conservation. To test this hypothesis, we have replaced Arabidopsis thaliana CENH3 with orthologs from other organisms to determine which sequences are essential for a functional CENH3. We have found that only the closely related CENH3 from Arabidopsis arenosa localized correctly while the others from distantly related species did not. Further experiments will test CENH3s from other closely related species, strengthening our knowledge about the properties of the centromere histone and its influence on chromosome segregations. INTRODUCTION The centromere is the locus on the chromosome where the kinetochore, which will be bound by the spindle microtubules, assembles. There are three broadly defined types of centromere DNA sequences : • • *Nature Reviews Neuroscience Normal histone H3 is highly conserved due to its importance in DNA packaging. In contrast, CENH3 primary sequence has evolved rapidly even between species with similar centromere structure. We hypothesize that there is a conserved structure that underlies CENH3 function, despite the lack of sequence conservation. GFP-CENH3/+ Selecting the organisms for this study Complementation Point and holocentric centromeres: Regional centromeres: O. sativa A. arenosa S. cerevisiae How far across evolutionary distance is function maintained? L. nivea C. elegans Can CENH3 that interacts with point and holocentric centromeres function in a regional centromere? S. cerevisiae (point) L. nivea (holocentric) C. elegans (holocentric) Z. mays O. sativa H. sapiens Native CENH3 Promoter The centromere contains the centromere specific histone (CENH3) which recruits kinetochore proteins. Seed Counting Assay of cenh3/+ O.sativa CENH3 /cenh3/+ Self-fertilizes Cloning strategy Regional -Mb of tandem repeats (A.thaliana & H.sapiens) Point -125 bp sequence (S.cerevisiae) Holocentric-microtubules bind along chromosome (C.elegans). Rapid evolution of CENH3 O. sativa genotyping results: complementation unlikely Test whether CENH3 orthologs localize correctly in Arabidopsis thaliana and complement a mutation in the endogenous CENH3 A. arenosa B. stricta B. holboellii What is the centromere? • METHODS % Lethality ABSTRACT CENH3 Terminator 6x Glycine 1/16=6.25% 1/4=25% Lethality only in cenh3 homozygote lacking GFP CENH3 Lethality in all cenh3 homozygote Plant Number Five siliques were collected from thirteen heterozygotes with the O. sativa GFP-tagged CENH3. Although two plants exhibited low percentage of lethality (15.5 & 17.7), it is not close enough to the expected percentage for complementation. CONCLUSION The correct localization of A. arenosa CENH3 and the non-localization of H. sapiens CENH3 suggest that a conserved structure exist only between closely related orthologs. Diffused fluorescence and seed counting assay of cenh3/+ with O. sativa suggest that complementation unlikely. pCAMBIA GFP CENH3 Variant Non-localization of S. cerevisiae and C. elegans CENH3s suggest that CENH3 from interact with holocentric and point FUTURE DIRECTIONS Continue with A. arenosa genotyping Agrobacterium in sol’n Transformed by dipping Hygromycin selection Image root tips RESULTS Fluorescent microscopy results A. thaliana (regional) A. arenosa (regional) C. elegans At this time, transformants are still too young to collect tissue from for genotyping. Homozygote mutants found in the T1, will be examined for any phenotypic variations from the wildtype. (holocentric) For CENH3 orthologs that lack localization: Are they expressed? • • RTPCR of CENH3 mRNA to determine if transgene was transcribed Western blot to determine if mRNA was expressed Further examination of rice CENH3: • (Figure above compares only half of the histone gene sequence from A.thaliana, A.arenosa, O.sativa, and H.sapiens) O. sativa H. sapiens S. cerevisiae (regional) (regional) (point) Progress of other orthologs: Cloning ongoing Boechera stricta, Boechera holboellii, Zea mays: and Brassica rapa (new on list): •A. thaliana with transgene •In process of cloning currently growing on ms plate Luzula nivea: •Construct will be sent to us A. thaliana CENH3 can be tagged with GFP Wild-type Genotype progeny from plant 5 and 13 for complementation cenh3/CENH3 REFERENCES A. arenosa showed fluorescence similar to A. thaliana CENH3, proving that the protein can correctly localize despite sequence differences. Wild-type cenh3 + GFP-CENH3 transgene Null mutation is embryo lethal allowing us to test complementation. Transgene with A.thaliana CENH3 showed kinetochore localization as well as complementation (plants had a wild-type phenotype). O. sativa CENH3 showed diffuse fluorescence within the nucleus, but localization in centromere is not clear. Possible over-expression or lack of protein degradation. No fluorescence shown with H. sapiens GFP-tagged CENH3. CENH3s from non-regional centromeres did not show fluorescence. Protein might be unrecognizable by the A.thaliana CENH3 loading machinery. 1) Cleveland, D.W., Mao, Y., and Sullivan, K.F. (2003). Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 112, 407-421. 2) Black, B.E., and Bassett, E.A. (2008). The histone variant CENP-A and centromere specification. Curr Opin Cell Biol 20, 91-100 4) Images were from: Napsal, CS Kuoh, Masur, ESA , BBC.co.uk ACKNOWLEDGEMENTS This research was supported in part by an NIH-IMSD award to UC Davis (GM-56765) and by a Howard Hughes Medical Institute grant (#52005892) in support of the Biology Undergraduate Scholars Program (BUSP) Special thanks to all the members of the Chan lab for their kindness and guidance. Thanks also to BUSP and the UC LEADS program for all of the support and mentorship.