Editing BioMicroCenter:Research (section)

== 2013 ==
===Copy number analysis in single cells - [http://web.mit.edu/biology/www/facultyareas/facresearch/amon.html AMON LAB]- [http://web.mit.edu/biology Biology] and [http://web.mit.edu/ki KI]===
Accurate segregation of chromosomes is critical for maintenance of genomic stability.   Failure to properly segregate chromosomes results in an unbalanced chromosome number, a condition known as aneuploidy.  Aneuploidy for chromosome 21 is the cause of Down Syndrome, and the majority of solid tumors harbor aneuploidies for one or more chromosomes.  Apart from these diseases, it has also been reported that normal, untransformed cells missegregate chromosomes as often as 20% of divisions (Carere et al., 1999; Shi & King, 2005).  Furthermore, multiple groups have reported even higher levels of aneuploidy in the normal mammalian brain, with over 20% of neurons and glia being aneuploidy (Faggioli, Wang, Vijg, & Montagna, 2012; Pack et al., 2005; Rehen et al., 2001; 2005).  Traditionally, these phenomena have been studied using spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH).  Unfortunately, these methods are susceptible to spreading and hybridization artifacts, respectively, and FISH is limited in the number of chromosomes that can be surveyed in a single experiment. The Amon laboratory has thus turned to single cell sequencing to provide a more robust and thorough assessment of aneuploidy in the brain and other tissues. <BR><BR>
Using tissue samples obtained from mice and humans, we isolated single cells by microaspiration and amplified genomic DNA using a commercially available kit.  We then relied on the BioMicro Center to prepare barcoded libraries and sequence these libraries using the Illumina HiSeq2000.  From sequencing reads, we were able to infer the copy number of each chromosome in a given cell.<BR><BR>

=== Cell cycle control in S. meliloti - [http://web.mit.edu/biology/www/facultyareas/facresearch/walker.html WALKER LAB] - [http://web.mit.edu/biology Biology], [http://web.mit.edu/cehs CEHS], and [http://web.mit.edu/ki KI]===
Proper regulation of the bacterial cell cycle is crucial for faithful replication and segregation of the genome. In some groups of bacteria, i.e. α-proteobacteria, tight regulation of cell cycle progression is also necessary for specific cellular differentiation required for adaptation to their diverse environmental niches. The symbiotic lifestyle of the soil bacterium, Sinorhizobium meliloti requires a drastic cellular differentiation that includes changes to the cell envelope, endoreduplication of the genome and loss of reproductive capacity. This differentiation requires a specific re-wiring of the S. meliloti cell cycle network to allow for the de-coupling of DNA replication and cell division. Although many of the well-characterized genes encoding the cell cycle regulatory network of the model α-proteobacterium, Caulobacter crescentus, are well conserved in S. meliloti, the function of these genes S. meliloti is poorly understood. It has been postulated that transcriptional control of cell cycle progression is a common and critical level of regulation in α-proteobacteria, but has only been demonstrated in C. crescentus. The BioMicro Center worked closely with Nicole De Nisco of the Walker lab work to perform global analysis of cell cycle regulated gene expression in S. meliloti, the first analysis of this kind conducted in an α-proteobacterium other than C. crescentus.<BR><BR>
Taking advantage of the S. meliloti cell synchronization method developed by Nicole De Nisco, the BioMicro Center and Nicole De Nisco utilized custom Agilent gene expression microarrays to measure gene expression in S. meliloti cells isolated during discrete cell cycle stages. Through statistical analysis and data clustering executed by the BioMicro Center informatics staff, the Walker lab was able to define a set of 462 genes with cell cycle transcripts in S. meliloti, confirming the conservation of cell cycle regulated gene expression between S. meliloti and C. crescentus and thereby expanding the paradigm of transcriptional control of α-proteobacterial cell cycle progression. Using BLAST and COG analysis performed by the BioMicro Center to compare the S. meliloti list to the list of 553 previously identified genes with cell cycle regulated transcripts in C. crescentus, the Walker Lab discovered a core set of 128 genes with conserved cell cycle dependent transcription between the two species completely independent of cell synchronization method.<BR><BR>
To determine which S. meliloti cell cycle regulated transcripts could be controlled by the transcriptional regulators CtrA and DnaA, the Walker Lab and BioMicro Center utilized previously described PWMs to search for CtrA and DnaA binding motifs within the list of 462 cell cycle regulated genes. This analysis revealed 64 genes with putative CtrA binding sites and 96 with putative DnaA binding sites. To provide support for the biological relevance of these sites, homologs of the S. meliloti genes identified in this analysis were found in 11 representative α-proteobacteria and then analyzed for conserved CtrA and DnaA binding motifs. The results of this analysis revealed a role for CtrA in regulation of cell division, flagellar biogenesis and coordination of DNA replication in S. meliloti, but by different strategies than previously defined in C. crescentus reflecting unique lifestyles and environmental niches of these two α-proteobacterial species. This lack of conservation of DnaA binding motifs in the homologs of S. meliloti genes demonstrating cell cycle regulated transcription revealed that the activity of DnaA as a transcriptional regulator in α-proteobacteria might be much less evolutionarily constrained than that of CtrA.