Andy Golden: Mentorship through the Years.

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Plug and play modular strategies for synthetic retrotransposons.

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.

Components of the spindle assembly checkpoint regulate the anaphase-promoting complex during meiosis in Caenorhabditis elegans.

Temperature-sensitive mutations in subunits of the Caenorhabditis elegans anaphase-promoting complex (APC) arrest at metaphase of meiosis I at the restrictive temperature. Embryos depleted of the APC co-activator FZY-1 by RNAi also arrest at this stage. To identify regulators and potential substrates of the APC, we performed a genetic suppressor screen with a weak allele of the APC subunit MAT-3/CDC23/APC8, whose defects are specific to meiosis. Twenty-seven suppressors that resulted in embryonic viability and larval development at the restrictive temperature were isolated. We have identified the molecular lesions in 18 of these suppressors, which correspond to five genes. In addition to a single intragenic suppressor, we found mutations in the APC co-activator fzy-1 and in three spindle assembly checkpoint genes, mdf-1, mdf-2, and mdf-3/san-1, orthologs of Mad1, Mad2, and Mad3, respectively. Reduction-of-function alleles of mdf-2 and mdf-3 suppress APC mutants and exhibit pleiotropic phenotypes in an otherwise wild-type background. Analysis of a single separation-of-function allele of mdf-1 suggests that MDF-1 has a dual role during development. These studies provide evidence that components of the spindle assembly checkpoint may regulate the metaphase-to-anaphase transition in the absence of spindle damage during C. elegans meiosis.

Multiple subunits of the Caenorhabditis elegans anaphase-promoting complex are required for chromosome segregation during meiosis I.

Two genes, originally identified in genetic screens for Caenorhabditis elegans mutants that arrest in metaphase of meiosis I, prove to encode subunits of the anaphase-promoting complex or cyclosome (APC/C). RNA interference studies reveal that these and other APC/C subunits are essential for the segregation of chromosomal homologs during meiosis I. Further, chromosome segregation during meiosis I requires APC/C functions in addition to the release of sister chromatid cohesion.

Active retrotransposition by a synthetic L1 element in mice.

Long interspersed element type 1 (L1) retrotransposons are ubiquitous mammalian mobile elements and potential tools for in vivo mutagenesis; however, native L1 elements are relatively inactive in mice when introduced as transgenes. We have previously described a synthetic L1 element, ORFeus, containing two synonymously recoded ORFs relative to mouse L1. It is significantly more active for retrotransposition in cell culture than all native L1 elements tested. To study its activity in vivo, we developed a transgenic mouse model in which ORFeus expression was controlled by a constitutive heterologous promoter, and we established definitive evidence for ORFeus retrotransposition activity both in germ line and somatic tissues. Germ line retrotransposition frequencies resulting in 0.33 insertions per animal are seen among progeny of ORFeus donor element heterozygotes derived from a single founder, representing a >20-fold increase over native L1 elements. We observe somatic transposition events in 100% of the ORFeus donor-containing animals, and an average of 17 different insertions are easily recovered from each animal; modeling suggests that the number of somatic insertions per animal exceeds this number by perhaps several orders of magnitude. Nearly 200 insertions were precisely mapped, and their distribution in the mouse genome appears random relative to transcription units and guanine-cytosine content. The results suggest that ORFeus may be developed into useful tools for in vivo mutagenesis.