Host Genome-Metagenome Analyses Using Combinatorial Network Methods Reveal Key Metagenomic and Host Genetic Features for Methane Emission and Feed Efficiency in Cattle.

Cattle production is one of the key contributors to global warming due to methane emission, which is a by-product of converting feed stuff into milk and meat for human consumption. Rumen hosts numerous microbial communities that are involved in the digestive process, leading to notable amounts of methane emission. The key factors underlying differences in methane emission between individual animals are due to, among other factors, both specific enrichments of certain microbial communities and host genetic factors that influence the microbial abundances.

Genetic-Metabolic Coupling for Targeted Metabolic Engineering.

Production of chemicals in microbes often employs potent biosynthetic enzymes, which can interact with the microbial native metabolism to affect cell fitness and product yield. However, production optimization largely relies on data collected from wild-type strains in the absence of metabolic perturbations, thus limiting their relevance to specific conditions. Here, we address this issue by coupling cell fitness to the production of thiamine diphosphate in Escherichia coli using a synthetic RNA biosensor.

Effects of genetic variation on the E. coli host-circuit interface.

Predictable operation of engineered biological circuitry requires the knowledge of host factors that compete or interfere with designed function. Here, we perform a detailed analysis of the interaction between constitutive expression from a test circuit and cell-growth properties in a subset of genetic variants of the bacterium Escherichia coli. Differences in generic cellular parameters such as ribosome availability and growth rate are the main determinants (89%) of strain-specific differences of circuit performance in laboratory-adapted strains but are responsible for only 35% of expression variation across 88 mutants of E.

Contextualizing context for synthetic biology--identifying causes of failure of synthetic biological systems.

Despite the efforts that bioengineers have exerted in designing and constructing biological processes that function according to a predetermined set of rules, their operation remains fundamentally circumstantial. The contextual situation in which molecules and single-celled or multi-cellular organisms find themselves shapes the way they interact, respond to the environment and process external information. Since the birth of the field, synthetic biologists have had to grapple with contextual issues, particularly when the molecular and genetic devices inexplicably fail to function as designed when tested in vivo.

Mammalian pre-mRNA 3' end processing factor CF I m 68 functions in mRNA export.

Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3' end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3' end processing complex, CF I(m)68, stimulates mRNA export.

Hierarchical inactivation of a synthetic human kinetochore by a chromatin modifier.

We previously used a human artificial chromosome (HAC) with a synthetic kinetochore that could be targeted with chromatin modifiers fused to tetracycline repressor to show that targeting of the transcriptional repressor tTS within kinetochore chromatin disrupts kinetochore structure and function. Here we show that the transcriptional corepressor KAP1, a downstream effector of the tTS, can also inactivate the kinetochore. The disruption of kinetochore structure by KAP1 subdomains does not simply result from loss of centromeric CENP-A nucleosomes.

Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line.

Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis.

Inactivation of a human kinetochore by specific targeting of chromatin modifiers.

We have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric alpha-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability.

A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes.

We describe a method for the isolation of conditional knockouts of essential multiply spliced genes in which the entire body of the gene downstream of the ATG start codon is left untouched but can be switched off rapidly and completely by adding tetracycline to the culture medium. The approach centers on a "promoter-hijack" strategy in which the gene's promoter is replaced with a minimal promoter responsive to the tetracycline-repressible transactivator (tTA). Elsewhere in the genome, a cloned fragment of the gene's promoter is used to drive expression of a tTA.

Subnuclear localization and dynamics of the Pre-mRNA 3' end processing factor mammalian cleavage factor I 68-kDa subunit.

Mammalian cleavage factor I (CF Im) is an essential factor that is required for the first step in pre-mRNA 3' end processing. Here, we characterize CF Im68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF Im68 accumulates in structures that partially overlap with nuclear speckles.

Distinct sequence motifs within the 68-kDa subunit of cleavage factor Im mediate RNA binding, protein-protein interactions, and subcellular localization.

Cleavage factor I(m) (CF I(m)) is required for the first step in pre-mRNA 3'-end processing and can be reconstituted in vitro from its heterologously expressed 25- and 68-kDa subunits. The binding of CF I(m) to the pre-mRNA is one of the earliest steps in the assembly of the cleavage and polyadenylation machinery and facilitates the recruitment of other processing factors. We identified regions in the subunits of CF I(m) involved in RNA binding, protein-protein interactions, and subcellular localization.