51 research outputs found
Pogostick: A New Versatile piggyBac Vector for Inducible Gene Over-Expression and Down-Regulation in Emerging Model Systems
Non-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as over-expression and ectopic expression of transgenes.We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www.addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals.This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles
Comparative Structural Analysis of Human DEAD-Box RNA Helicases
DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members
Earth: Atmospheric Evolution of a Habitable Planet
Our present-day atmosphere is often used as an analog for potentially
habitable exoplanets, but Earth's atmosphere has changed dramatically
throughout its 4.5 billion year history. For example, molecular oxygen is
abundant in the atmosphere today but was absent on the early Earth. Meanwhile,
the physical and chemical evolution of Earth's atmosphere has also resulted in
major swings in surface temperature, at times resulting in extreme glaciation
or warm greenhouse climates. Despite this dynamic and occasionally dramatic
history, the Earth has been persistently habitable--and, in fact,
inhabited--for roughly 4 billion years. Understanding Earth's momentous changes
and its enduring habitability is essential as a guide to the diversity of
habitable planetary environments that may exist beyond our solar system and for
ultimately recognizing spectroscopic fingerprints of life elsewhere in the
Universe. Here, we review long-term trends in the composition of Earth's
atmosphere as it relates to both planetary habitability and inhabitation. We
focus on gases that may serve as habitability markers (CO2, N2) or
biosignatures (CH4, O2), especially as related to the redox evolution of the
atmosphere and the coupled evolution of Earth's climate system. We emphasize
that in the search for Earth-like planets we must be mindful that the example
provided by the modern atmosphere merely represents a single snapshot of
Earth's long-term evolution. In exploring the many former states of our own
planet, we emphasize Earth's atmospheric evolution during the Archean,
Proterozoic, and Phanerozoic eons, but we conclude with a brief discussion of
potential atmospheric trajectories into the distant future, many millions to
billions of years from now. All of these 'Alternative Earth' scenarios provide
insight to the potential diversity of Earth-like, habitable, and inhabited
worlds.Comment: 34 pages, 4 figures, 4 tables. Review chapter to appear in Handbook
of Exoplanet
Polychaete fauna associated with holdfasts of the large brown alga Himantothallus grandifolius in Admiralty Bay, King George Island, Antarctic
The Splicing History of an mRNA Affects Its Level of Translation and Sensitivity to Cleavage by the Virion Host Shutoff Endonuclease during Herpes Simplex Virus Infections
Synergistic heterozygosity for TGFβ1 SNPs and BMPR2 mutations modulates the age at diagnosis and penetrance of familial pulmonary arterial hypertension
PYM binds the cytoplasmic exon-junction complex and ribosomes to enhance translation of spliced mRNAs
The structure of the ASAP core complex reveals the existence of a Pinin-containing PSAP complex
- …