Faculty Bibliography

It has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones. How mitotic progression and quality control might be influenced by histones is less well characterized. We show that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Prior to anaphase, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of Sgo1p. Reinstating the pericentric enrichment of Sgo1p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint.

GeneDesign is a set of web applications that provides public access to a nucleotide manipulation pipeline for synthetic biology. The server is public and freely accessible, and the source is available for download under the New BSD License. Since GeneDesign was published and made publicly available 3 years ago, we have made its code base more efficient, added several algorithms and modules, updated the restriction enzyme library, added batch processing capabilities, and added several command line modules, all of which we briefly describe here.

A major challenge in undergraduate life science curricula is the continual evaluation and development of courses that reflect the constantly shifting face of contemporary biological research. Synthetic biology offers an excellent framework within which students may participate in cutting-edge interdisciplinary research and is therefore an attractive addition to the undergraduate biology curriculum. This new discipline offers the promise of a deeper understanding of gene function, gene order, and chromosome structure through the de novo synthesis of genetic information, much as synthetic approaches informed organic chemistry. While considerable progress has been achieved in the synthesis of entire viral and prokaryotic genomes, fabrication of eukaryotic genomes requires synthesis on a scale that is orders of magnitude higher. These high-throughput but labor-intensive projects serve as an ideal way to introduce undergraduates to hands-on synthetic biology research. We are pursuing synthesis of Saccharomyces cerevisiae chromosomes in an undergraduate laboratory setting, the Build-a-Genome course, thereby exposing students to the engineering of biology on a genomewide scale while focusing on a limited region of the genome. A synthetic chromosome III sequence was designed, ordered from commercial suppliers in the form of oligonucleotides, and subsequently assembled by students into approximately 750-bp fragments. Once trained in assembly of such DNA "building blocks" by PCR, the students accomplish high-yield gene synthesis, becoming not only technically proficient but also constructively critical and capable of adapting their protocols as independent researchers. Regular "lab meeting" sessions help prepare them for future roles in laboratory science.

Yeast and mammalian genomes are replete with nearly identical copies of long dispersed repeats in the form of retrotransposons. Mechanisms clearly exist to maintain genome structure in the face of potential rearrangement between the dispersed repeats, but the nature of this machinery is poorly understood. Here we describe a series of distinct "retrotransposon overdose" (RO) lineages in which the number of Ty1 elements in the Saccharomyces cerevisiae genome has been increased by as much as 10 fold. Although these RO strains are remarkably normal in growth rate, they demonstrate an intrinsic supersensitivity to DNA-damaging agents. We describe the identification of mutants in the DNA replication pathway that enhance this RO-specific DNA damage supersensitivity by promoting ectopic recombination between Ty1 elements. Abrogation of normal DNA replication leads to rampant genome instability primarily in the form of chromosomal aberrations and confirms the central role of DNA replication accuracy in the stabilization of repetitive DNA.

Recent progress in L1 biology highlights its role as a major driving force in the evolution of mammalian genome structure and function. This coincides with direct confirmation of the preponderance of long interspersed elements in mammalian genomes at the nucleotide level by large scale sequencing efforts. Two assay systems have been prominently featured in L1 studies over the past decade, which are used to assess L1 activities in cultured cells and transgenic mice respectively. However, constructing retrotransposon assay vectors and subsequent mapping of integration sites remain technically challenging aspects of the field. Synthetic biology approaches have changed the playing field with regard to the strategic design of retrotransposons. To streamline the construction and optimization of synthetic retrotransposons, we have implemented a highly efficient modular design for L1 vectors allowing "plug and play" swapping of individual modules as new knowledge is gained and optimization of constructs proceeds. Seven functional modules are divided by strategically placed unique restriction sites. These are utilized to facilitate module exchange and construction of L1 vectors for gene targeting, transgenesis and cell culture assays. A "double SfiI" strategy utilizing two non-complementary overhangs allows insert swapping to be carried out with a single, robust restriction/ligation cycle. The double-SfiI strategy is generic and can be applied to many other problems in synthetic biology or genetic engineering. To facilitate genomic mapping of L1 insertions, we have developed an optimized inverse PCR protocol using 4-base cutters and step-down cycling conditions. Using this protocol, de novo L1 insertions can be efficiently recovered after a single round of PCR. The proposed modular design also incorporates features allowing streamlined insertion mapping without repeated optimization. Furthermore, we have presented evidence that efficient L1 retrotransposition is not dependent on pCEP4 conferred autonomous replication capabilities when a shortened puromycin selection protocol is used, providing a great opportunity for further optimization of L1 cell culture assay vectors by using alternative vector backbones.