In 2005 and 2006, two of our undergraduate lab members presented their research at the State of North Carolina Undergraduate Research Symposium,
as listed below.

2006:

Student Author(s):	Anderson, Marybeth
Dept & College or University:	Biology, UNC-Chapel Hill
Research Mentor(s):	Elaine Yeh/Biology, University of North Carolina at Chapel Hill
Title of Presentation:	The Dependence of Kinetochore Complexes on Centromere Binding

Correct mitotic segregation of chromosomes relies upon the attachment of the microtubule to the chromosome at the kinetochore complex. Unlike in other organisms, the attachment of microtubules to the yeast chromosomes requires only one microtubule per kinetochore; furthermore, each kinetochore is composed of at least nine protein complexes. Of these major complexes CBF3, containing the essential protein Ndc10, is located adjacent to the nucleosome and is important in formation of the kinetochore. This procedure tested whether known kinetochore proteins Ctf19 and Nuf2 localized to the kinetochore in an Ndc10 dependent manner. By comparing the intensity of GFP tagged Ctf19 in strains with and without Ndc10 it was determined that Ctf19, a member of the COMA complex, failed to assemble at the kinetochore in the absence of Ndc10. Nuf2, a member of the Ndc80 complex, was visualized as distinct foci along the spindle equator in the absence of Ndc10, however various abnormalities in the localization of Nuf2 were observed. We are currently looking at additional kinetochore complexes to determine their stability in the absence of kinetochore attachment.

Student Author(s):	Bond, Lisa M.
Dept & College or University:	Biology, UNC-Chapel Hill
Research Mentor(s):	Kerry Bloom/Biology, UNC-Chapel Hill
Title of Presentation:	Cohesin Is a Stable Component of Pericentric Chromatin

The biorientation of joined sister chromatids at metaphase permits the accurate transfer of genetic material to daughter cells during cell division. The cohesin complex, composed of Mcd1p/Scc1p, Scc3p, Smc1p, and Smc3p, facilitates accurate transfer by holding sister chromatids together until its cleavage at anaphase onset. This complex forms a cylindrical array around the mitotic spindle in the budding yeast Saccharomyces cerevisiae. A paradox is raised by the fact that 3-5 times more cohesin is bound at pericentric chromatin than along chromosome arms, but sister centromeres are separated by 600-800 nm prior to cohesin cleavage (Pearson et al, 2001). A recently proposed model accounts for cohesin enrichment at pericentric chromatin and sister centromere separation by suggesting that individual chromatids fold back upon themselves at each centromere to form “c-loops,” and that cohesin is distributed not only along the pericentric regions of juxtaposed sister chromatid arms (interstrand cohesin), but also along the overlapping regions of individual, folded chromatids (interstrand cohesin) (Bloom et al., 2006.). With this model in mind, this study focused on determining the stability of pericentric cohesin. Fluorescence Recovery after Photobleaching (FRAP) was used to monitor cohesin stability in cells expressing Smc3p-GFP. FRAP of histone H2B-GFP served as a positive control. Analysis revealed that cohesin fluorescence recovered above the background in only 2 of 12 cells, while histone H2B was dynamic in 5 out of 5 cells analyzed. These results suggest that cohesin is stably bound to pericentric chromatin. The stability of this structure may be important for establishing sister chromatid biorientation and/or contributing to stability of the mitotic spindle. Further use of FRAP analysis to ascertain the nature of specific forms of cohesin will contribute to the overall determination of the nature of pericentric cohesin.

2005:

Student Author(s):	Bond, Lisa M.
Dept & College or University:	Biology, UNC-Chapel Hill
Research Mentor(s):	Kerry Bloom/Biology, UNC-Chapel Hill
Title of Presentation:	Examination of the Role of the Cohesin Complex in Sister Chromatid Cohesion

 
Accurate transfer of a single copy of the chromosome complement of a dividing cell to each of its daughter cells relies on the proper alignment of joined sister chromatids along the metaphase plate and the subsequent separation and segregation of these chromatids.  Recent genetic research suggests that the cohesin complex, which consists of MCD1/SCC1, SCC3, SMC1, and SMC3, plays an integral role in the joining of sister chromatids, and that the breakdown of this complex at the onset of anaphase permits chromatid separation.  This study compared kinetochore distribution in a temperature sensitive mcd1-1 mutant of the yeast Saccharomyces cerevisiae to wildtype, through the observation of a component of the kinetochore (CSE4) fused to green fluorescent protein (GFP).  Preliminary analysis of the acquired data indicates that the behavior of sister chromatids during mitosis in mcd1-1 mutants is similar to sister chromatid behavior during mitosis in wildtype cells.  These data suggest that the cohesin complex is not the only mechanism of joining sister chromatids, as all cohesion between sister chromatids is not lost in the mcd1-1 mutant.  These mutants will continue to be observed for a careful analysis of their phenotype, and the precise location and characteristics of the cohesin complex in mitosis will be further studied using SMC3-GFP.