Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins. The machinery that initiates DNA synthesis is highly conserved, but the sites where the replication initiation proteins bind have diverged significantly. Functional comparative genomics is an obvious approach to study the evolution of replication origins. However, to date, the Saccharomyces cerevisiae replication origin map is the only genome map available. Using an iterative approach that combines computational prediction and functional validation, we have generated a high-resolution genome-wide map of DNA replication origins in Kluyveromyces lactis. Unlike other yeasts or metazoans, K. lactis autonomously replicating sequences (KlARSs) contain a 50 bp consensus motif suggestive of a dimeric structure. This motif is necessary and largely sufficient for initiation and was used to dependably identify 145 of the up to 156 non-repetitive intergenic ARSs projected for the K. lactis genome. Though similar in genome sizes, K. lactis has half as many ARSs as its distant relative S. cerevisiae. Comparative genomic analysis shows that ARSs in K. lactis and S. cerevisiae preferentially localize to non-syntenic intergenic regions, linking ARSs with loci of accelerated evolutionary change.

Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase ␣ in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Pol␣. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Pol␣ without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Pol␣ at the elongating replication fork-first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.The key players of the replication machinery are the DNA polymerases that synthesize the leading and lagging daughter strands and the replicative helicase that unwinds the parental strands ahead of the polymerases. Coordination between the helicase and the polymerases is critical during replication. Uncoupling of these two molecular machines, especially during lagging strand synthesis, may result in an unrestrained helicase and the exposure of extensive single-stranded DNA (ssDNA), as observed in checkpoint mutants treated with hydroxyurea (HU) (37). Although there is no direct evidence, the implication is that the replicative helicase would be moving at a faster pace than would the DNA polymerase if synchrony were destroyed. In Escherichia coli, the replicative helicase (DnaB) and the primase (DnaG) are coupled by direct contact to form a tight complex (3). In T7, processivity of the gp5 polymerase in lagging strand synthesis requires coupling to the gp4 helicase (16). Recent studies of the budding yeast Saccharomyces cerevisiae suggest that Mrc1 may couple DNA polymerase ε and the MCM helicase on the leading strand as well as activate the checkpoint response under replication stress (1,22,28). A candidate for coupling DNA polymerase ␣ primase and the MCM helicase on the lagging strand is Mcm10, because Mcm10 interacts with subunits of the Mcm2-7 helicase (26, 29) as well as Pol␣ (14, 33) and the stability of Pol␣ requires Mcm10 in both budding yeast and human cells (8,33). Mcm10 is an essential protein known to be involved in various aspects of the replication process. It is required during both initiation and elongation steps of DNA replication and interacts with a wide range of replication factors, such as ORC (17,23,29), MCM helicase, DNA polymerases ε and ␦ (23), Cdc45 (...

The intervertebral discs, located between adjacent vertebrae, are required for stability of the spine and distributing mechanical load throughout the vertebral column. All cell types located in thes middle regions of the discs, called nuclei pulposi, are derived from the embryonic notochord. Recently, it was shown that the hedgehog signaling pathway plays an essential role during formation of nuclei pulposi. However, during the time that nuclei pulposi are forming, Shh is expressed in both the notochord and the nearby floor plate. To determine the source of SHH protein sufficient for formation of nuclei pulposi we removed Shh from either the floor plate or the notochord using tamoxifen-inducible Cre alleles. Removal of Shh from the floor plate resulted in phenotypically normal intervertebral discs, indicating that Shh expression in this tissue is not required for disc patterning. In addition, embryos that lacked Shh in the floor plate had normal vertebral columns, demonstrating that Shh expression in the notochord is sufficient for pattering the entire vertebral column. Removal of Shh from the notochord resulted in the absence of Shh in the floor plate, loss of intervertebral discs and vertebral structures. These data indicate that Shh expression in the notochord is sufficient for patterning of the intervertebral discs and the vertebral column.

BackgroundGrain size is one of the key factors determining yield and quality in rice. A large number of genes are involved in the regulation of grain size parameters such as grain length and grain width. Different alleles of these genes have different impacts on the grain size traits under their control. However, the combined influence of multiple alleles of different genes on grain size remains to be investigated. Six key genes known to influence grain size were investigated in this study: GS3, GS5, GS6, GW2, qSW5/GW5, and GW8/OsSPL16. Allele and grain measurement data were used to develop a regression equation model that can be used for molecular breeding of rice with desired grain characteristics.ResultsA total of 215 diverse rice germplasms, which originated from or were developed in 28 rice-consuming countries, were used in this study. Genotyping analysis demonstrated that a relatively small number of allele combinations were preserved in the diverse population and that these allele combinations were significantly associated with differences in grain size. Furthermore, in several cases, variation at a single gene was sufficient to influence grain size, even when the alleles of other genes remained constant. The data were used to develop a regression equation model for prediction of rice grain size, and this was tested using data from a further 34 germplasms. The model was significantly correlated with three of the four grain size-related traits examined in this study.ConclusionRice grain size is strongly influenced by specific combinations of alleles from six different genes. A regression equation model developed from allele and grain measurement data can be used in rice breeding programs for the development of new rice varieties with desired grain size and shape.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-015-0066-1) contains supplementary material, which is available to authorized users.