Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae

Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix–loop–helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5′-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.     

Cbf14 copy number variation in the A, B, and D genomes of diploid and polyploid wheat

Freezing tolerance and winter hardiness are complex traits. In the Triticeae, two loci on the group 5 chromosome homoeologs are repeatedly identified as having major effects on these traits. Recently, we found that segments of the genomic region at one of these loci, Frost resistance-2 (Fr-2) is copy number variable in barley. Freezing-tolerant winter-hardy genotypes have greater tandem copy numbers of the genomic region encompassing the C-repeat binding factor genes Cbf2A and Cbf4B at Fr-H2 than the less freezing-tolerant nonwinter-hardy genotypes. Here we report that in wheat the Cbf14 gene at Fr-2 is copy number variable. Using DNA blot hybridizations, we estimated copy numbers of Cbf14 across the different genomes of diploid and polyploid wheat. Copy numbers of Cbf14 are lower in the B genome than in the A and D genomes across all ploidy levels. Among hexaploid red wheats, winter genotypes harbor greater Cbf14 copy numbers than spring genotypes. Cbf14 copy numbers also vary across the red winter wheats such that hard wheats harbor greater copy numbers than soft wheats. Analysis of hexaploid wheat chromosome 5 substitution lines indicates that Cbf14 copy numbers in the introgressions are stable in the different backgrounds. Taken together our data suggest that higher copy number states existed in the diploid wild ancestors prior to the polyploidization events and that the loss of Cbf14 copies occurred in the cultivated germplasm.     

Reciprocal Signaling between the Ectoderm and a Mesendodermal Left-Right Organizer Directs Left-Right Determination in the Sea Urchin Embryo

During echinoderm development, expression of nodal on the right side plays a crucial role in positioning of the rudiment on the left side, but the mechanisms that restrict nodal expression to the right side are not known. Here we show that establishment of left-right asymmetry in the sea urchin embryo relies on reciprocal signaling between the ectoderm and a left-right organizer located in the endomesoderm. FGF/ERK and BMP2/4 signaling are required to initiate nodal expression in this organizer, while Delta/Notch signaling is required to suppress formation of this organizer on the left side of the archenteron. Furthermore, we report that the H⁺/K⁺-ATPase is critically required in the Notch signaling pathway upstream of the S3 cleavage of Notch. Our results identify several novel players and key early steps responsible for initiation, restriction, and propagation of left-right asymmetry during embryogenesis of a non-chordate deuterostome and uncover a functional link between the H⁺/K⁺-ATPase and the Notch signaling pathway.