Faculty Bibliography

We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.

Long noncoding RNAs (lncRNAs), a recently discovered class of cellular RNAs, play important roles in the regulation of many cellular developmental processes. Although lncRNAs have been systematically identified in various systems, most of them have not been functionally characterized in vivo in animal models. In this study, we identified 128 testis-specific Drosophila lncRNAs and knocked out 105 of them using an optimized three-component CRISPR/Cas9 system. Among the lncRNA knockouts, 33 (31%) exhibited a partial or complete loss of male fertility, accompanied by visual developmental defects in late spermatogenesis. In addition, six knockouts were fully or partially rescued by transgenes in a trans configuration, indicating that those lncRNAs primarily work in trans Furthermore, gene expression profiles for five lncRNA mutants revealed that testis-specific lncRNAs regulate global gene expression, orchestrating late male germ cell differentiation. Compared with coding genes, the testis-specific lncRNAs evolved much faster. Moreover, lncRNAs of greater functional importance exhibited higher sequence conservation, suggesting that they are under constant evolutionary selection. Collectively, our results reveal critical functions of rapidly evolving testis-specific lncRNAs in late Drosophila spermatogenesis.

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts.

The truncated glucocorticoid receptor mutant gene 465* codes for a protein that is interrupted by a frame-shift mutation in the second zinc finger of the natural DNA binding domain. Thus, 465* represents the natural amino acid sequence 1-465 followed by 21 novel amino acids starting at position 466. The entire ligand binding domain is missing. Prior studies have shown that transient transfection of the glucocorticoid-resistant leukemic T-cell clone ICR-27 with a plasmid expressing 465* rapidly reduces the number of viable cells. This response does not require activation by a steroid, and a hybrid protein consisting of green fluorescent protein fused to 465* is found primarily in the cytoplasm. In the present study, we present evidence that the decrease in cell number is due to a form of cell death that bears many of the classic characteristics of apoptosis. Expression of the 465* protein can be detected a few hours after electroporation and is followed by activation of caspase-3 as well as reduction of the mitochondrial inner transmembrane potential. The caspase-3 inhibitor ZVAD-fmk blocks 465*-dependent cell death when added acutely after electroporation, but fails to do so later. We conclude that the novel 465* gene causes cell death by apoptosis.